Methods for inhibiting oxidative modification of proteins

ABSTRACT

The instant invention provides compositions and methods for modeling post-Amadori AGE formation and the identification and characterization of effective inhibitors of post-Amadori AGE formation, and such identified inhibitor compositions. The instant invention also teaches new methods to treat or prevent oxidative modification of proteins, including low density lipoproteins, to treat or prevent lipid peroxidation, and to treat or prevent atherosclerosis, comprising administering an amount effective of one of the compounds of the invention to treat or prevent the disorder.

CROSS REFERENCE

This application is a continuation-in-part of U.S. patent applications,Ser. No. 60/103,795 filed Oct. 9, 1998; Ser. No. 08/971,285 filed Nov.17, 1997 now U.S. Pat. No. 6,228,858 and Ser. No. 08/711,555, filed Sep.10, 1996, now U.S. Pat. No. 5,985,857, and claims priority to U.S.Provisional Application for Patent Ser. No. 60/003,628, filed Sep. 12,1995, the contents of each of which are hereby incorporated by referencein their entirety.

STATEMENT OF GOVERNMENT RIGHTS

Some of the work disclosed has been supported in part by NIH Grant DK43507 and NIH grant DK-19971, therefore, the United States Governmentmay have certain rights in the invention.

BACKGROUND OF THE INVENTION

The instant invention is in the field of Advanced Glycation End-products(AGEs), their formation, detection, identification, inhibition, andinhibitors thereof.

Protein Aging and Advanced Glycosylation End-products

Nonenzymatic glycation by glucose and other reducing sugars is animportant post-translational modification of proteins that has beenincreasingly implicated in diverse pathologies. Irreversiblenonenzymatic glycation and crosslinking through a slow, glucose-inducedprocess may mediate many of the complications associated with diabetes.Chronic hyperglycemia associated with diabetes can cause chronic tissuedamage which can lead to complications such as retinopathy, nephropathy,and atherosclerotic disease. (Cohen and Ziyadeh, 1996, J. Amer. Soc.Nephrol. 7:183-190). It has been shown that the resulting chronic tissuedamage associated with long-term diabetes mellitus arise in part from insitu immune complex formation by accumulated immunoglobulins and/orantigens bound to long-lived structural proteins that have undergoneAdvanced Glycosylation End-product (AGE) formation, via non-enzymaticglycosylation (Brownlee et al., 1983, J. Exp. Med. 158:1739-1744). Theprimary protein target is thought to be extra-cellular matrix associatedcollagen. Nonenzymatic glycation of proteins, lipids, and nucleic acidsmay play an important role in the natural processes of aging. Recentlyprotein advanced glycation has been associated with β-amyloid depositsand formation of neurofibrillary tangles in Alzheimer disease, andpossibly other neurodegenerative diseases involving amyloidosis (Colacoand Harrington, 1994, NeuroReport 5: 859-861). Glycated proteins havealso been shown to be toxic, antigenic, and capable of triggeringcellular injury responses after uptake by specific cellular receptors(see for example, Vlassara, Bucala & Striker, 1994, Lab. Invest.70:138-151; Vlassara et al., 1994, PNAS(USA) 91:11704-11708; Daniels &Hauser, 1992, Diabetes 41:1415-1421; Brownlee, 1994, Diabetes43:836-841; Cohen et al., 1994, Kidney Int. 45:1673-1679; Brett et al.,1993, Am. J. Path. 143:1699-1712; and Yan et al., 1994, PNAS(USA)91:7787-7791).

The appearance of brown pigments during the cooking of food is auniversally recognized phenomenon, the chemistry of which was firstdescribed by Maillard in 1912, and which has subsequently led toresearch into the concept of protein aging. It is known that stored andheat-treated foods undergo nonenzymatic browning that is characterizedby crosslinked proteins which decreases their bioavailibility. It wasfound that this Maillard reaction occurred in vivo as well, when it wasfound that a glucose was attached via an Amadori rearrangement to theamino-terminal of the α-chain of hemoglobin.

The instant disclosure teaches previously unknown, and unpredictedmechanism of formation of post-Amadori advanced glycation end products(Maillard products; AGEs) and methods for identifying and characterizingeffective inhibitors of post-Amadori AGE formation. The instantdisclosure demonstrates the unique isolation and kineticcharacterization of a reactive protein intermediate competent in formingpost-Amadori AGEs, and for the first time teaching methods which allowfor the specific elucidation and rapid quantitative kinetic study of“late” stages of the protein glycation reaction.

In contrast to such “late” AGE formation, the “early” steps of theglycation reaction have been relatively well characterized andidentified for several proteins (Harding, 1985, Adv. Protein Chem.37:248-334; Monnier & Baynes eds., 1989, The Maillard Reaction in Aging,Diabetes, and Nutrition (Alan R. Liss, New York); Finot et al., 1990,eds. The Maillard Reaction in Food Processing, Human Nutrition andPhysiology (Birkhauser Verlag, Basel)). Glycation reactions are known tobe initiated by reversible Schiff-base (aldimine or ketimine) additionreactions with lysine side-chain ε-amino and terminal α-amino groups,followed by essentially irreversible Amadori rearrangements to yieldketoamine products e.g. 1-amino-1-deoxy-ketoses from the reaction ofaldoses (Baynes et al., 1989, in The Maillard Reaction in Aging,Diabetes, and Nutrition, ed. Monnier and Baynes, (Alan R. Liss, NewYork, pp 43-67). Typically, sugars initially react in their open-chain(not the predominant pyranose and furanose structures) aldehydo or ketoforms with lysine side chain ε-amino and terminal α-amino groups throughreversible Schiff base condensation (Scheme I). The resulting aldimineor ketimine products then undergo Amadori rearrangements to giveketoamine Amadori products, i.e. 1-amino-1-deoxy-ketoses from thereaction of aldoses (Means & Chang, 1982, Diabetes 31, Suppl. 3:1-4;Harding, 1985, Adv. Protein Chem. 37:248-334). These Amadori productsthen undergo, over a period of weeks and months, slow and irreversibleMaillard “browning” reactions, forming fluorescent and other productsvia rearrangement, dehydration, oxidative fragmentation, andcross-linking reactions. These post-Amadori reactions, (slow Maillard“browning” reactions), lead to poorly characterized Advanced GlycationEnd-products (AGEs).

As with Amadori and other glycation intermediaries, free glucose itselfcan undergo oxidative reactions that lead to the production of peroxideand highly reactive fragments like the dicarbonyls glyoxal andglycoaldehyde. Thus the elucidation of the mechanism of formation of avariety of AGEs has been extremely complex since most in vitro studieshave been carried out at extremely high sugar concentrations.

In contrast to the relatively well characterized formation of these“early” products, there has been a clear lack of understanding of themechanisms of forming the “late” Maillard products produced inpost-Amadori reactions, because of their heterogeneity, long reactiontimes, and complexity. The lack of detailed information about thechemistry of the “late” Maillard reaction stimulated research toidentify fluorescent AGE chromophores derived from the reaction ofglucose with amino groups of polypeptides. One such chromophore,2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) was identified afternonenzymatic browning of bovine serum albumin and polylysine withglucose, and postulated to be representative of the chromophore presentin the intact polypeptides. (Pongor et al., 1984, PNAS(USA)81:2684-2688). Later studies established FFI to be an artifact formedduring acid hydrolysis for analysis.

A series of U.S. Patents have issued in the area of inhibition ofprotein glycosylation and cross-linking of protein sugar amines basedupon the premise that the mechanism of such glycosylation andcross-linking occurs via saturated glycosylation and subsequentcross-linking of protein sugar amines via a single basic, and repeatingreaction. These patents include U.S. Pat. Nos. 4,665,192; 5,017,696;4,758,853; 4,908,446; 4,983,604; 5,140,048; 5,130,337; 5,262,152;5,130,324; 5,272,165; 5,221,683; 5,258,381; 5,106,877; 5,128,360;5,100,919; 5,254,593; 5,137,916; 5,272,176; 5,175,192; 5,218,001;5,238,963; 5,358,960; 5,318,982; and 5,334,617. (All U.S. Patents citedare hereby incorporated by reference in their entirety).

The focus of these U.S. Patents, are a method for inhibition of AGEformation focused on the carbonyl moiety of the early glycosylationAmadori product, and in particular the most effective inhibitiondemonstrated teaches the use of exogenously administered aminoguanidine.The effectiveness of aminoguanidine as an inhibitor of AGE formation iscurrently being tested in clinical trials.

Inhibition of AGE formation has utility in the areas of, for example,food spoilage, animal protein aging, and personal hygiene such ascombating the browning of teeth. Some notable, though quantitativelyminor, advanced glycation end-products are pentosidine andN^(ε)-carboxymethyllysine (Sell and Monnier, 1989, J. Biol. Chem.264:21597-21602; Ahmed et al., 1986, J. Biol. Chem. 261:4889-4894).

The Amadori intermediary product and subsequent post-Amadori AGEformation, as taught by the instant invention, is not fully inhibited byreaction with aminoguanidine. Thus, the formation of post-Amadori AGEsas taught by the instant disclosure occurs via an important and uniquereaction pathway that has not been previously shown, or even previouslybeen possible to demonstrate in isolation. It is a highly desirable goalto have an efficient and effective method for identifying andcharacterizing effective post-Amadori AGE inhibitors of this “late”reaction. By providing efficient screening methods and model systems,combinatorial chemistry can be employed to screen candidate compoundseffectively, and thereby greatly reducing time, cost, and effort in theeventual validation of inhibitor compounds. It would be very useful tohave in vivo methods for modeling and studying the effects ofpost-Amadori AGE formation which would then allow for the efficientcharacterization of effective inhibitors.

Inhibitory compounds that are biodegradable and/or naturally metabolizedare more desirable for use as therapeutics than highly reactivecompounds which may have toxic side effects, such as aminoguanidine.

SUMMARY OF THE INVENTION

In accordance with the present invention, a stable post-Amadori advancedglycation end-product (AGE) precursor has been identified which can thenbe used to rapidly complete the post-Amadori conversion intopost-Amadori AGEs. This stable product is a presumed sugar saturatedAmadori/Schiff base product produced by the further reaction of theearly stage protein/sugar Amadori product with more sugar. In apreferred embodiment, this post-Amadori/Schiff base intermediary hasbeen generated by the reaction of target protein with ribose sugar.

The instant invention provides for a method of generating stableprotein-sugar AGE formation intermediary precursors via a novel methodof high sugar inhibition. In a preferred embodiment the sugar used isribose.

The instant invention provides for a method for identifying an effectiveinhibitor of the formation of late Maillard products comprising:generating stable protein-sugar post-Amadori advanced glycationend-product intermediates by incubating a protein with sugar at asufficient concentration and for sufficient length of time to generatestable post-Amadori AGE intermediates; contacting said stableprotein-sugar post-Amadori advanced glycation end-product intermediateswith an inhibitor candidate; identifying effective inhibition bymonitoring the formation of post-Amadori AGEs after release of thestable protein-sugar post-Amadori advanced glycation end-productintermediates from sugar induced equilibrium. Appropriate sugarsinclude, and are not limited to ribose, lyxose, xylose, and arabinose.It is believed that certain conditions will also allow for use ofglucose and other sugars. In a preferred embodiment the sugar used isribose.

The instant invention teaches that an effective inhibitor ofpost-Amadori AGE formation via “late” reactions can be identified andcharacterized by the ability to inhibit the formation of post-AmadoriAGE endproducts in an assay comprising; generating stable protein-sugarpost-Amadori advanced glycation end-product intermediates by incubatinga protein with sugar at a sufficient concentration and for sufficientlength of time to generate stable post-Amadori AGE intermediates;contacting said stable protein-sugar post-Amadori advanced glycationend-product intermediates with an inhibitor candidate; identifyingeffective inhibition by monitoring the formation of post-Amadori AGEsafter release of the stable protein-sugar post-Amadori advancedglycation end-product intermediates from sugar induced equilibrium. In apreferred embodiment the assay uses ribose.

Thus the methods of the instant invention allow for the rapid screeningof candidate post-Amadori AGE formation inhibitors for effectiveness,greatly reducing the cost and amount of work required for thedevelopment of effective small molecule inhibitors of post-Amadori AGEformation. The instant invention teaches that effective inhibitors ofpost-Amadori “late” reactions of AGE formation include derivatives ofvitamin B₆ and vitamin B₁, in the preferred embodiment the specificspecies being pyridoxamine and thiamine pyrophosphate.

The instant invention teaches new methods for rapidly inducing diabeteslike pathologies in rats comprising administering ribose to the subjectanimal. Further provided for is the use of identified inhibitorspyridoxamine and thiamine pyrophosphate in vivo to inhibit post-AmadoriAGE induced pathologies.

The present invention encompasses compounds for use in the inhibition ofAGE formation and post-Amadori AGE pathologies, and pharmaceuticalcompositions

containing such compounds of the general formula:

Formula I

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group; and salts thereof.

The present invention also encompasses compounds of the general formula

Formula II

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH

R₂ and R₆ is H, OH, SH, NH₂, C 1-18 alkyl, alkoxy or alkene;

R₄ and R₅ are H, C 1-18 alkyl, alkoxy or alkene;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group, and salts thereof

In a preferred embodiment at least one of R₄, R₅ and R₆ are H.

In addition, the instant invention also envisions compounds of theformulas

The compounds of the present invention can embody one or more electronwithdrawing groups, such as and not limited to —NH₂, —NHR, —NR₂, —OH,—OCH₃, —OCR, and —NH—COCH₃ where R is C 1-6 alkyl.

The instant invention encompasses pharmaceutical compositions whichcomprise one or more of the compounds of the present invention, or saltsthereof, in a suitable carrier. The instant invention encompassesmethods for administering pharmaceuticals of the present invention fortherapeutic intervention of pathologies which are related to AGEformation in vivo. In one preferred embodiment of the present inventionthe AGE related pathology to be treated is related to diabeticnephropathy.

The instant invention also teaches new methods to treat or preventoxidative modification of proteins, including low density lipoproteins,to treat or prevent lipid peroxidation, and to treat or preventatherosclerosis, comprising administering an amount effective of one ofthe compounds of the invention to treat or prevent the disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation in bovine serum albumin (BSA). FIG. 1APyrdoxamine (PM); FIG. 1B pyridoxal phosphate (PLP); FIG. 1C pyridoxal(PL); FIG. 1D pyridoxine (PN).

FIG. 2 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation in bovine serumalbumin. FIG. 2A Thiamine pyrophosphate (TPP); FIG. 2B thiaminemonophosphate (TP); FIG. 2C thiamine (T); FIG. 2D aminoguanidine (AG).

FIG. 3 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation in human methemoglobin (Hb). FIG. 3APyridoxamine (PM); FIG. 3B pyridoxal phosphate (PLP); FIG. 3C pyridoxal(PL); FIG. 3D pyridoxine (PN).

FIG. 4 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation in humanmethemoglobin. FIG. 2A Thiamine pyrophosphate (TPP); FIG. 2B thiaminemonophosphate (TP); FIG. 2C thiamine (T); FIG. 2D aminoguanidine (AG).

FIG. 5 is a bar graph comparison of the inhibition of the glycation ofribonuclease A by thiamine pyrophosphate (TPP), pyridoxamine (PM) andaminoguanidine (AG).

FIG. 6A is a graph of the kinetics of glycation of RNase A (10 mg/mL) byribose as monitored by ELISA. FIG. 6B is a graph showing the dependenceof reciprocal half-times on ribose concentration at pH 7.5.

FIG. 7 are two graphs showing a comparison of uninterrupted andinterrupted glycation of RNase by glucose (7B) and ribose (7A), asdetected by ELISA.

FIG. 8 are two graphs showing kinetics of pentosidine fluorescence(arbitrary units) increase during uninterrupted and interrupted riboseglycation of RNase. FIG. 8A Uninterrupted glycation in the presence of0.05 M ribose. FIG. 8B Interrupted glycation after 8 and 24 hours ofincubation.

FIG. 9 is a graph which shows the kinetics of reactive intermediatebuildup.

FIG. 10 are graphs of Post-Amadori inhibition of AGE formation byribose. FIG. 10A graphs data where aliquots were diluted into inhibitorcontaining buffers at time 0. FIG. 10B graphs data where samples wereinterrupted at 24 h, and then diluted into inhibitor containing buffers.

FIG. 11 is a graph showing dependence of the initial rate of formationof antigenic AGE on pH following interruption of glycation.

FIG. 12 are two graphs showing the effect of pH jump on ELISA detectedAGE formation after interrupted glycation. Interrupted samples left 12days at 37° C. in pH 5.0 buffer produced substantial AGEs (33%; FIG.12B) when pH was changed to 7.5, as compared to the normal controlsample not exposed to low pH (FIG. 12A).

FIG. 13 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation during uninterrupted glycation ofribonuclease A (RNase A) by ribose. FIG. 13A Pyridoxamine (PM); FIG. 13Bpyridoxal-5′-phosphate (PLP); FIG. 13C pyridoxal (PL); FIG. 13Dpyridoxine (PN).

FIG. 14 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation duringuninterrupted glycation of ribonuclease A (RNase A) by ribose. FIG. 14AThiamine pyrophosphate (TPP); FIG. 14B thiamine monophosphate (TP); FIG.14C thiamine (T); FIG. 14D aminoguanidine (AG).

FIG. 15 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation during uninterrupted glycation of bovineserum albumin (BSA) by ribose. FIG. 15A Pyridoxamine (PM); FIG. 15Bpyridoxal-5′-phosphate (PLP); FIG. 15C pyridoxal (PL); FIG. 15Dpyridoxine (PN).

FIG. 16 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation duringuninterrupted glycation of bovine serum albumin (BSA) by ribose. FIG.16A Thiamine pyrophosphate (TPP); FIG. 16B thiamine monophosphate (TP);FIG. 16C thiamine (T); FIG. 16D aminoguanidine (AG).

FIG. 17 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation during uninterrupted glycation of humanmethemoglobin (Hb) by ribose. FIG. 17A Pyridoxamine (PM); FIG. 17Bpyridoxal-5′-phosphate (PLP); FIG. 17C pyridoxal (PL); FIG. 17Dpyridoxine (PN).

FIG. 18 is a series of graphs depicting the effect of vitamin B₆derivatives on post-Amadori AGE formation after interrupted glycation byribose. FIG. 18A BSA and Pyridoxamine (PM); FIG. 18B BSA andpyridoxal-5′-phosphate (PLP); FIG. 18C BSA and pyridoxal (PL); FIG. 18DRNase and pyridoxamine (PM).

FIG. 19 are graphs depicting the effect of thiamine pyrophosphate onpost-Amadori AGE formation after interrupted glycation by ribose. FIG.19A RNase, FIG. 19B BSA.

FIG. 20 are graphs depicting the effect of aminoguanidine onpost-Amadori AGE formation after interrupted glycation by ribose. FIG.20A RNase, FIG. 20B BSA.

FIG. 21 is a graph depicting the effect of N-acetyl-L-lysine onpost-Amadori AGE formation after interrupted glycation by ribose.

FIG. 22 are bar graphs showing a comparison of post-Amadori inhibitionof AGE formation by thiamine pyrophosphate (TPP), pyridoxamine (PM) andaminoguanidine (AG) after interrupted glycation of RNase (FIG. 22A) andBSA (FIG. 22B) by ribose.

FIG. 23 is a bar graph showing the effects of Ribose treatment in vivoalone on rat tail-cuff blood pressure. Treatment was with 0.05 M, 0.30M, and 1 M Ribose (R) injected for 1, 2 or 8 Days (D).

FIG. 24 is a bar graph showing the effects of Ribose treatment in vivoalone on rat creatinine clearance (Clearance per 100 g Body Weight).Treatment was with 0.05 M, 0.30 M, and 1 M Ribose (R) injected for 1, 2or 8 Days (D).

FIG. 25 is a bar graph showing the effects of Ribose treatment in vivoalone on rat Albuminuria (Albumin effusion rate). Treatment was with0.30 M, and 1 M Ribose (R) injected for 1, 2 or 8 Days (D).

FIG. 26 is a bar graph showing the effects of inhibitor treatment invivo, with or without ribose, on rat tail-cuff blood pressure. Treatmentgroups were: 25 mg/kg body weight aminoguanidine (AG); 25 or 250 mg/kgbody weight Pyridoxamine (P); 250 mg/kg body weight Thiaminepyrophosphate (T), or with 1 M Ribose (R).

FIG. 27 is a bar graph showing the effects of inhibitor treatment invivo, with or without ribose, on rat creatinine clearance (Clearance per100 g body weight). Treatment groups were: 25 mg/kg body weightaminoguanidine (AG); 25 or 250 mg/kg body weight Pyridoxamine (P); 250mg/kg body weight Thiamine pyrophosphate (T), or with 1 M Ribose (R).

FIG. 28 is a bar graph showing the effects of inhibitor treatment invivo without ribose, and ribose alone on rat Albuminuria (Albumineffusion rate). Treatment groups were: 25 mg/kg body weightaminoguanidine (AG); 250 mg/kg body weight Pyridoxamine (P); 250 mg/kgbody weight Thiamine pyrophosphate (T), or treatment with 1 M Ribose (R)for 8 days (D). Control group had no treatment.

FIG. 29 is a bar graph showing the effects of inhibitor treatment invivo, with 1 M ribose , on rat Albuminuria (Albumin effusion rate).Treatment groups were: 25 mg/kg body weight aminoguanidine (AG); 25 and250 mg/kg body weight Pyridoxamine (P); 250 mg/kg body weight Thiaminepyrophosphate (T), or treatment with 1 M Ribose (R) for 8 days (D)alone. Control group had no treatment.

FIG. 30A depicts Scheme 1 showing a diagram of AGE formation fromprotein. FIG. 30B depicts Scheme 2, a chemical structure ofaminoguanidine. FIG. 30C depicts Scheme 3, chemical structures forthiamine, thiamine-5′-phosphate, and thiamine pyrophosphate. FIG. 30Ddepicts Scheme 4, chemical structures of pyridoxine, pyridoxamine,pyridoxal-5′-phosphate, and pyridoxal. FIG. 30E depicts Scheme 5,kinetics representation of AGE formation. FIG. 30F depicts Scheme 6,kinetics representation of AGE formation and intermediate formation.

FIGS. 31(A-B) shows a 125 MHz C-13 NMR Resonance spectrum ofRiobonuclease Amadori Intermediate prepared by 24 HR reaction with 99%[2-C13]Ribose.

FIGS. 32(A-B) are graphs which show AGE intermediary formation using thepentoses Xylose, Lyxose, Arabinose and Ribose.

FIG. 33 is a graph showing the results of glomeruli staining at pH 2.5with Alcian blue.

FIG. 34 is a graph showing the results of glomeruli staining at pH 1.0with Alcian blue.

FIG. 35 is a graph showing the results of immunofluroescent glomerulistaining for RSA.

FIG. 36 is a graph showing the results of immunofluroescent glomerulistaining for Heparan Sulfate Proteoglycan Core protein.

FIG. 37 is a graph showing the results of immunofluroescent glomerulistaining for Heparan Sulfate Proteoglycan side-chain.

FIG. 38 is a graph showing the results of analysis of glomeruli sectionsfor average glomerular volume.

FIG. 39. Structure and mass spectrum of the TFAME derivative of lysine.Lysine was derivatized to its methyl ester using methanolic-HCl thenconverted to its TFAME derivative with trifluoroacetic anhydride. The180 ion (base peak) was used for quantification of lysine by GC/MS.

FIG. 40. Structure of derivatized CML and CEL. CML and CEL werederivatized to TFAMEs as described for lysine. Ions 392 and 379 wereused for quantification of CML and CEL, respectively.

FIG. 41. Mass Spectrum of derivatized CML and CEL. CML and CEL werederivatized to TFAMEs as described for lysine. Ions 392 and 379 wereused for quantification of CML and CEL, respectively.

FIG. 42. Structure of MDA-lysine (A) and HNE-lysine (B). MDA-lysine andHNE-lysine were derivatized to TFAMEs as described for lysine. Ions 474and 323 were used for quantification of MDA-lysine and HNE-lysine,respectively.

FIG. 43. Mass Spectrum of MDA-lysine (A) and HNE-lysine (B). MDA-lysineand HNE-lysine were derivatized to TFAMEs as described for lysine. Ions474 and 323 were used for quantification of MDA-lysine and HNE-lysine,respectively.

FIG. 44. Loss of free amino groups during reaction of PM with PUFAs. PM(1 mM) was incubated alone (τ) or in the presence of oleate (λ),linoleate (σ), or arachidonate (ν) (5 mM) in 0.2 M sodium phosphatebuffer pH 7.4 for 6 days at 37° C.

Aliquots were withdrawn at various time intervals and analyzed for freeamino groups using the TNBS assay. Data shown are the mean and standarddeviation of 3 independent experiments.

FIG. 45. LC-MS analysis of a PM/linoleate reaction at day 6. PM (1 mM)was reacted with linoleate (5 mM). The day 6 sample was analyzed byLC-MS as described in Materials and Methods. Panel A and panel Brepresent the mass spectra taken from the major products.

FIG. 46. Kinetics of formation of products 267, 339, and 305. PM (1 mM)was reacted with linoleate (5 mM) as described previously. The sampleswere analyzed by RP-HPLC using absorbance detection at 294 nm. Panel Ashows a chromatogram of a day 6 reaction and the formation of products339, 267 and 305. The products were quantified (Panel B) based on theirarea ratios to the internal standard PL (267 (λ), 339 (ν), and 305 (σ)).The data shown are the mean and standard deviation from 3 independentexperiments.

FIG. 47. Proposed structure of products 267 and 339. Products 267 and339 were hypothesized to be hexamide and nonanedioic acid amidederivatives of PM.

FIG. 48. TNBS reactivity of synthetic 339. An estimated 80 nmoles ofsynthetic 339 was analyzed by the TNBS assay. An equivalent amount of PMstandard was analyzed for comparison.

FIG. 49. Proposed mechanism to the formation of 267 and 339. Oxidationof linoleic acid proceeds through formation of linoleic acid 9- and13-lipid hydroperoxides. These may then dehydrate to form ketoacids.Nucleophilic attack of PM on the ketoacids could lead to formation ofthe amide derivatives via oxidative cleavage of the Schiff base.

FIG. 50. Preferential cleavage of the Schiff base to form the amidederivatives may be driven by radical stabilization within the conjugatedsystem. The carbinolamine, precursor of the Schiff base adduct, wouldthen undergo hydrogen abstraction. The resulting radical might bestabilized by the conjugated carbon system, directing the cleavagereaction to form the 6-carbon amide derivative. The same mechanism wouldapply to formation of the nonanedioic acid amide derivative.

FIG. 51. Proposed structure for product 305. Though completecharacterization of 305 was not accomplished, a molecular formulaobtained by high resolution FAB-MS suggests the product is either apyrrole or furan derivative of PM.

FIG. 52. Inhibition of CML and CEL formation by PM during reactions ofRNase with arachidonate. RNase (1 mM (10 mM lysine)) was reacted witharachidonate (100 mM) alone (□) or in the presence of 1 mM PM (▪) in 200mM sodium phosphate buffer, pH 7.4 at 37° C. for 6 days. At various timepoints 1 mg of protein was removed and prepared for GC/MS analysis. CMLand CEL were analyzed by GC/MS as their trifluoroacetyl methyl esters.Data shown is the average and range of two independent experiments.

FIG. 53. Inhibition of MDA-lysine and HNE-lysine by PM during reactionsof RNase with arachidonate. RNase (1 mM) was reacted with arachidonate(100 mM) alone (□) or in the presence of 1 mM PM (▪) in 200 mM sodiumphosphate buffer, pH 7.4 at 37° C. for 6 days. At various time intervals1 mg of protein was removed and prepared for GC/MS analysis. MDA-lysineand HNE-lysine were analyzed by GC/MS as their trifluoroacetyl methylesters. Data shown is the average and range of two independentexperiments.

FIG. 54. Prolongation of conjugated diene formation by PM during coppercatalyzed oxidation of LDL. LDL (50 μg/ml) was incubated alone (▪) oroxidized in the presence of 5 μM Cu²⁺ () or with 5 μM Cu²⁺ in thepresence of 100 μM (▴) or 250 μM (Δ) PM. Conjugated diene formation wasmonitored at an absorbance of 234 nm. Results are shown for twoindependent pools of LDL (A and B).

FIGS. 55(A-B). PM inhibits formation of CML and CEL duringcopper-catalyzed oxidation of LDL. LDL (50 μg/ml) was incubated for 3hours alone or oxidized with 5 μM Cu²⁺ in the presence of 10 or 100 μMPM in PBS. Approximately 1 mg of protein was removed at 3 hours andreduced with 500 mM NaBH₄ (Requena, 1997). Following reduction, thesamples were dialyzed, delipidated and hydrolyzed in 6N HCl. CML and CELwere analyzed as their trifluoroacetyl methyl esters. Results shownrepresent the average and range of two independent pools of LDL.

FIGS. 56(A-B). PM inhibits formation of MDA-lysine and HNE-lysine duringcopper catalyzed oxidation of LDL. LDL (50 μg/ml) was incubated for 3hours alone or oxidized with 5 μM Cu²⁺ in the presence of or 100 μM PMin PBS. Approximately 1 mg of protein was removed at 3 hours and reducedwith 500 mM NaBH₄. Following reduction, the samples were dialyzed,delipidated and hydrolyzed in 6N HCl. MDA-lysine and HFM-lysine wereanalyzed as their trifluoroacetyl methyl esters. Results represent theaverage and range of two independent pools of LDL.

FIG. 57. PM prevents lysine modification during copper catalyzedoxidation of LDL. LDL (50 μg/ml) was incubated for 3 hours alone oroxidized with 5 μM Cu²⁺ in the presence of 100 or 250 μM PM in PBS.Approximately 150 μg of protein was removed at 3 hours and immediatelyhydrolyzed, or reduced with NaBH₄ then hydrolyzed, in 6N HCl for 24hours at 110° C. Following hydrolysis the samples were reconstituted inPickering buffer A and analyzed by cation-exchange HPLC. The resultsshown for both non-reduced and reduced lysine are the average values +/−the range from two independent pools of LDL.

FIG. 58. Effect of PM on TBARs formation during oxidation of linoleate.Linoleate was oxidized alone (5 mM) (▪) or in the presence of PM (1 mM)() for 6 days at 37° C. in sodium phosphate buffer, pH 7.4. Aliquotswere removed at various time intervals and analyzed for TBARs asdescribed above. Data shown represents the mean +/− standard deviationof 3 independent experiments.

FIG. 59. Effect of PM on the rate of linoleate oxidation. Linoleate (5mM) was oxidized alone (▪) or in the presence of PM (1 mM) () for 6days. Linoleate was extracted using acidified chloroform:methanol(Folch, 1957). Following addition of the internal standard, palmitate,the samples were derivatized with borontrichloride-methanol and thefatty acids analyzed as their methyl esters by GC/MS. Quantification wasbased on standard curves generated from linoleate and palmitatestandards. Data shown is the mean +/− standard deviation of 3independent experiments.

FIG. 60. Mechanism by which PM may act as an antioxidant. PM (A) maydonate a hydrogen to radicals produced either during the initiation orpropagation phases of lipid peroxidation. The resulting PM radical (B)is stabilized by the aromatic ring system (C).

DETAILED DESCRIPTION

Animal Models for Protein Aging

Alloxan induced diabetic Lewis rats have been used as a model forprotein aging to demonstrate the in vivo effectiveness of inhibitors ofAGE formation. The correlation being demonstrated is between inhibitionof late diabetes related pathology and effective inhibition of AGEformation (Brownlee, Cerami, and Vlassara, 1988, New Eng. J. Med.318(20):1315-1321). Streptozotocin induction of diabetes in Lewis rats,New Zealand White rabbits with induced diabetes, and geneticallydiabetic BB/Worcester rats have also been utilized, as described in, forexample, U.S. Pat. No. 5,334,617 (incorporated by reference). A majorproblem with these model systems is the long time period required todemonstrate AGE related injury, and thus to test compounds for AGEinhibition. For example, 16 weeks of treatment was required for the ratstudies described in U.S. Pat. No. 5,334,617, and 12 weeks for therabbit studies. Thus it would be highly desirable and useful to have amodel system for AGE related diabetic pathology that will manifest in ashorter time period, allowing for more efficient and expeditiousdetermination of AGE related injury and the effectiveness of inhibitorsof post-Amadori AGE formation.

Antibodies to AGEs

An important tool for studying AGE formation is the use of polyclonaland monoclonal antibodies that are specific for AGEs elicited by thereaction of several sugars with a variety of target proteins. Theantibodies are screened for resultant specificity for AGEs that isindependent of the nature of the protein component of the AGE (Nakayamaet al., 1989, Biochem. Biophys. Res. Comm. 162: 740-745; Nakayama etal., 1991, J. Immunol. Methods 140: 119-125; Horiuchi et al., 1991, JBiol. Chem. 266: 7329-7332; Araki et al., 1992, J. Biol. Chem. 267:10211-10214; Makita et al., 1992, J. Biol. Chem. 267: 5133-5138). Suchantibodies have been used to monitor AGE formation in vivo and in vitro.

Thiamine—Vitamin B₁

The first member of the Vitamin B complex to be identified, thiamine ispractically devoid of pharmacodynamic actions when given in usualtherapeutic doses; and even large doses were not known to have anyeffects. Thiamine pyrophosphate is the physiologically active form ofthiamine, and it functions mainly in carbohydrate metabolism as acoenzyme in the decarboxylation of α-keto acids. Tablets of thiaminehydrochloride are available in amounts ranging from 5 to 500 mg each.Thiamine hydrochloride injection solutions are available which contain100 to 200 mg/ml.

For treating thiamine deficiency, intravenous doses of as high as 100mg/L of parenteral fluid are commonly used, with the typical dose of 50to 100 mg being administered. GI absorption of thiamine is believed tobe limited to 8 to 15 mg per day, but may be exceed by oraladministration in divided doses with food.

Repeated administration of glucose may precipitate thiamine deficiencyin under nourished patients, and this has been noted during thecorrection of hyperglycemia.

Surprisingly, the instant invention has found, as shown by in vitrotesting, that administration of thiamine pyrophosphate at levels abovewhat is normally found in the human body or administered for dietarytherapy, is an effective inhibitor of post-Amadori antigenic AGEformation, and that this inhibition is more complete than that possibleby the administration of aminoguanidine.

Pyridoxine—Vitamin B₆

Vitamin B₆ is typically available in the form of pyridoxinehydrochloride in over-the-counter preparations available from manysources. For example Beach pharmaceuticals Beelith Tablets contain 25 mgof pyridoxine hydrochloride that is equivalent to 20 mg of B₆, otherpreparations include Marlyn Heath Care Marlyn Formula 50 which contain 1mg of pyridoxine HCl and Marlyn Formula 50 Mega Forte which contains 6mg of pyridoxine HCl, Wyeth-Ayerst Stuart Prenatal® tablets whichcontain 2.6 mg pyridoxine HCl, J&J-Merck Corp. Stuart Formula® tabletscontain 2 mg of pyridoxine HCl, and the CIBA Consumer Sunkist Children'schewable multivitamins which contain 1.05 mg of pyridoxine HCl, 150% ofthe U.S. RDA for children 2 to 4 years of age, and 53% of the U.S. RDAfor children over 4 years of age and adults. (Physician's Desk Referencefor nonprescription drugs, 14th edition (Medical Economics DataProduction Co., Montvale, N.J., 1993).

There are three related forms of pyridoxine, which differ in the natureof the substitution on the carbon atom in position 4 of the pyridinenucleus: pyridoxine is a primary alcohol, pyridoxal is the correspondingaldehyde, and pyridoxamine contains an aminomethyl group at thisposition. Each of these three forms can be utilized by mammals afterconversion by the liver into pyridoxal-5′-phosphate, the active form ofthe vitamin. It has long been believed that these three forms areequivalent in biological properties, and have been treated as equivalentforms of vitamin B₆ by the art. The Council on Pharmacy and Chemistryhas assigned the name pyridoxine to the vitamin.

The most active antimetabolite to pyridoxine is 4-deoxypyridoxine, forwhich the antimetabolite activity has been attributed to the formationin vivo of 4-deoxypyridoxine-5-phosphate, a competitive inhibitor ofseveral pyridoxal phosphate-dependent enzymes. The pharmacologicalactions of pyridoxine are limited, as it elicits no outstandingpharmacodynamic actions after either oral or intravenous administration,and it has low acute toxicity, being water soluble. It has beensuggested that neurotoxicity may develop after prolonged ingestion of aslittle as 200 mg of pyridoxine per day. Physiologically, as a coenzyme,pyridoxine phosphate is involved in several metabolic transformations ofamino acids including decarboxylation, transamination, and racemization,as well as in enzymatic steps in the metabolism of sulfur-containing andhydroxy-amino acids. In the case of transamination, pyridoxal phosphateis aminated to pyridoxamine phosphate by the donor amino acid, and thebound pyridoxamine phosphate is then deaminated to pyridoxal phosphateby the acceptor α-keto acid. Thus vitamin B complex is known to be anecessary dietary supplement involved in specific breakdown of aminoacids. For a general review of the vitamin B complex see ThePharmacological Basis of Therapeutics, 8th edition, ed. Gilman, Rall,Nies, and Taylor (Pergamon Press, New York, 1990, pp. 1293-4; pp.1523-1540).

Surprisingly, the instant invention has discovered that effectivedosages of the metabolically transitory pyridoxal amine form of vitaminB₆ (pyridoxamine), at levels above what is normally found in the humanbody, is an effective inhibitor of post-Amadori antigenic AGE formation,and that this inhibition may be more complete than that possible by theadministration of aminoguanidine.

Formation of Stable Amadori/Schiff Base Intermediary

The typical study of the reaction of a protein with glucose to form AGEshas been by ELISA using antibodies directed towards antigenic AGEs, andthe detection of the production of an acid-stable fluorescent AGE,pentosidine, by HPLC following acid hydrolysis. Glycation of targetproteins (i.e. BSA or RNase A) with glucose and ribose were compared bymonitoring ELISA reactivity of polyclonal rabbit anti-Glucose-AGE-RNaseand anti-Glucose-AGE-BSA antibodies. The antigen was generated byreacting 1 M glucose with RNase for 60 days and BSA for 90 days. Theantibodies (R618 and R479) were screened and showed reactivity with onlyAGEs and not the protein, except for the carrier immunogen BSA.

EXAMPLE 1 Thiamine Pyrophosphate and Pyridoxamine Inhibit the Formationof Antigenic Advanced Glycation End-products From Glucose: ComparisonWith Aminoguanidine

Some B₆ vitamers, especially pyridoxal phosphate (PLP), have beenpreviously proposed to act as “competitive inhibitors” of earlyglycation, since as aldehydes they themselves can form Schiff basesadducts with protein amino groups (Khatami et al., 1988, Life Sciences43:1725-1731) and thus limit the amount of amines available for glucoseattachment. However, effectiveness in limiting initial sugar attachmentis not a predictor of inhibition of the conversion of any Amadoriproducts formed to AGEs. The instant invention describes inhibitors of“late” glycation reactions as indicated by their effects on the in vitroformation of antigenic AGEs (Booth et al., 1996, Biochem. Biophys. Res.Com. 220:113-119).

Chemicals

Bovine pancreatic ribonuclease A (RNase) was chromatographically pure,aggregate-free grade from Worthington Biochemicals. Bovine Serum albumin(BSA; fraction V, fatty-acid free), human methemoglobin (Hb), D-glucose,pyridoxine, pyridoxal, pyridoxal 5′phosphate, pyridoxamine, thiamine,thiamine monophosphate, thiamine pyrophosphate, and goat alkalinephosphatase-conjugated anti-rabbit IgG were all from Sigma Chemicals.Aminoguanidine hydrochloride was purchased from Aldrich Chemicals.

Uninterrupted Glycation With Glucose

Bovine serum albumin, ribonuclease A, and human hemoglobin wereincubated with glucose at 37° C. in 0.4 M sodium phosphate buffer of pH7.5 containing 0.02% sodium azide. The protein, glucose (at 1.0 M), andprospective inhibitors (at 0.5, 3, 15 and 50 mM) were introduced intothe incubation mixture simultaneously. Solutions were kept in the darkin capped tubes. Aliquots were taken and immediately frozen untilanalyzed by ELISA at the conclusion of the reaction. The incubationswere for 3 weeks (Hb) or 6 weeks (RNase, BSA).

Preparation of Polyclonal Antibodies to AGE Proteins

Immunogen preparation followed earlier protocols (Nakayama et al., 1989,Biochem. Biophys. Res. Comm. 162:740-745; Horiuchi et al., 1991, J.Biol. Chem. 266:7329-7332; Makita et al., 1992, J. Biol. Chem.267:5133-5138). Briefly, immunogen was prepared by glycation of BSA(R479 antibodies) or RNase (R618 antibodies) at 1.6 g protein in 15 mlfor 60-90 days using 1.5 M glucose in 0.4 M sodium phosphate buffer ofpH 7.5 containing 0.05% sodium azide at pH 7.4 and 37° C. New Zealandwhite rabbit males of 8-12 weeks were immunized by subcutaneousadministration of a 1 ml solution containing 1 mg/ml of glycated proteinin Freund's adjuvant. The primary injection used the complete adjuvantand three boosters were made at three week intervals with Freund'sincomplete adjuvant. Rabbits were bled three weeks after the lastbooster. The serum was collected by centrifugation of clotted wholeblood. The antibodies are AGE-specific, being unreactive with eithernative proteins (except for the carrier) or with Amadori intermediates.The polyclonal anti-AGE antibodies have proven to be a sensitive andvaluable analytical tool for the study of “late” AGE formation in vitroand in vivo. The nature of the dominant antigenic AGE epitope or haptenremains in doubt, although recently it has been proposed that theprotein glycoxidation product carboxymethyl lysine (CML) may be adominant antigen of some antibodies (Reddy et al., 1995, Biochem.34:10872-10878). Earlier studies have failed to reveal ELISA reactivitywith model CmL compounds (Makita et al., 1992, J. Biol. Chem.267:5133-5138).

ELISA Detection of AGE Products

The general method of Engvall (1981, Methods Enzymol. 70:419-439) wasused to perform the ELISA. Typically, glycated protein samples werediluted to approximately 1.5 ug/ml in 0.1 M sodium carbonate buffer ofpH 9.5 to 9.7. The protein was coated overnight at room temperature onto96-well polystyrene plates by pippetting 200 ul of the protein solutionin each well (0.3 ug/well). After coating, the protein was washed fromthe wells with a saline solution containing 0.05% Tween-20. The wellswere then blocked with 200 ul of 1% casein in carbonate buffer for 2 hat 37° C. followed by washing. Rabbit anti-AGE antibodies were dilutedat a titer of about 1:350 in incubation buffer, and incubated for 1 h at37° C., followed by washing. In order to minimize background readings,antibodies R479 used to detect glycated RNase were raised againstglycated BSA, and antibodies R618 used to detect glycated BSA andglycated Hb were raised against glycated RNase. An alkalinephosphatase-conjugated antibody to rabbit IgG was then added as thesecondary antibody at a titer of 1:2000 or 1:2500 (depending on lot) andincubated for 1 h at 37° C., followed by washing. Thep-nitrophenylphosphate substrate solution was then added (200 ul/well)to the plates, with the absorbance of the released p-nitrophenolatebeing monitored at 410 nm with a Dynatech MR 4000 microplate reader.

Controls containing unmodified protein were routinely included, andtheir readings were subtracted, the corrections usually beingnegligible. The validity of the use of the ELISA method inquantitatively studying the kinetics of AGE formation depends on thelinearity of the assay (Kemeny & Challacombe, 1988, ELISA and OtherSolid Phase Immunoassays, John Wiley & Sons, Chichester, U.K.). Controlexperiments were carried out, for example, demonstrating that the linearrange for RNase is below a coating concentration of about 0.2-0.3mg/well.

Results

FIGS. 1A-D are graphs which show the effect of vitamin B₆ derivatives onpost-Amadori AGE formation in bovine serum albumin glycated withglucose. BSA (10 mg/ml) was incubated with 1.0 M glucose in the presenceand absence of the various indicated derivative in 0.4 M sodiumphosphate buffer of pH 7.5 at 37° C. for 6 weeks. Aliquots were assayedby ELISA using R618 anti-AGE antibodies. Concentrations of theinhibitors were 3, 15 and 50 mM. Inhibitors used in FIGS. (1A)Pyridoxamine (PM); (1B) pyridoxal phosphate (PLP); (1C) pyridoxal (PL);(1D) pyridoxine (PN).

FIG. 1 (control curves) demonstrates that reaction of BSA with 1.0 Mglucose is slow and incomplete after 40 days, even at the high sugarconcentration used to accelerate the reaction. The simultaneousinclusion of different concentrations of various B₆ vitamers markedlyaffects the formation of antigenic AGEs. (FIGS. 1A-D) Pyridoxamine andpyridoxal phosphate strongly suppressed antigenic AGE formation at eventhe lowest concentrations tested, while pyridoxal was effective above 15mM. Pyridoxine was slightly effective at the highest concentrationstested.

FIGS. 2A-D are graphs which show the effect of vitamin B₁ derivativesand aminoguanidine (AG) on AGE formation in bovine serum albumin. BSA(10 mg/ml) was incubated with 1.0 M glucose in the presence and absenceof the various indicated derivative in 0.4 M sodium phosphate buffer ofpH 7.5 at 37° C. for 6 weeks. Aliquots were assayed by ELISA using R618anti-AGE antibodies. Concentrations of the inhibitors were 3, 15 and 50mM. Inhibitors used in FIGS. (2A) Thiamine pyrophosphate (TPP); (2B)thiamine monophosphate (TP); (2C) thiamine (T); (2D) aminoguanidine(AG).

Of the various B₁ vitamers similarly tested (FIGS. 2A-D), thiaminepyrophosphate was effective at all concentrations tested (FIG. 2C),whereas thiamine and thiamine monophosphate were not inhibitory. Mostsignificantly it is remarkable to note the decrease in the final levelsof AGEs formed observed with thiamine pyrophosphate, pyridoxal phosphateand pyridoxamine. Aminoguanidine (FIG. 2D) produced some inhibition ofAGE formation in BSA, but less so than the above compounds. Similarstudies were carried out with human methemaglobin and bovineribonuclease A.

FIGS. 3A-D are graphs which show the effect of vitamin B₆ derivatives onAGE formation in human methemoglobin. Hb (1 mg/ml) was incubated with1.0 M glucose in the presence and absence of the various indicatedderivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37° C. for 3weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies.Concentrations of the inhibitors were 0.5, 3, 15 and 50 mM. Inhibitorsused in FIGS. (3A) Pyridoxamine (PM); (3B) pyridoxal phosphate (PLP);(3C) pyridoxal (PL); (3D) pyridoxine (PN).

It had been previously reported that Hb of a diabetic patient contains acomponent that binds to anti-AGE antibodies, and it was proposed thatthis glycated Hb (termed Hb-AGE, not to be confused with Hb_(Alc)) couldbe useful in measuring long-term exposure to glucose. The in vitroincubation of Hb with glucose produces antigenic AGEs at an apparentlyfaster rate than observed with BSA. Nevertheless, the different B₆(FIGS. 3A-D) and B₁ (FIGS. 4A-C) vitamers exhibited the same inhibitiontrends in Hb, with pyridoxamine and thiamine pyrophosphate being themost effective inhibitors in each of their respective families.Significantly, in Hb, aminoguanidine only inhibited the rate of AGEformation, and not the final levels of AGE formed (FIG. 4D).

With RNase the rate of antigenic AGE formation by glucose wasintermediate between that of Hb and BSA, but the extent of inhibitionwithin each vitamer series was maintained. Again pyridoxamine andthiamine pyrophosphate were more effective that aminoguanidine (FIG. 5).

FIGS. 4A-D are graphs which show the effect of vitamin B₁ derivativesand aminoguanidine (AG) on AGE formation in human methemoglobin. Hb (1mg/ml) was incubated with 1.0 M glucose in the presence and absence ofthe various indicated derivative in 0.4 M sodium phosphate buffer of pH7.5 at 37° C. for 3 weeks. Aliquots were assayed by ELISA using R618anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3, 15and 50 mM. Inhibitors used in FIGS. (4A) Thiamine pyrophosphate (TPP);(4B) thiamine monophosphate (TP); (4C) thiamine (T); (4D) aminoguanidine(AG).

FIG. 5 is a bar graph which shows a comparison of the inhibition of theglycation of ribonuclease A by thiamine pyrophosphate (TPP),pyridoxamine (PM) and aminoguanidine (AG). RNase (1 mg/ml) was incubatedwith 1.0 M glucose (glc) in the presence and absence of the variousindicated derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37°C. for 6 weeks. Aliquots were assayed by ELISA using R479 anti-AGEantibodies. The indicated percent inhibition was computed from ELISAreadings in the absence and presence of the inhibitors at the 6 weektime point. Concentrations e inhibitors were 0.5, 3, 15 and 50 mM.

Discussion

These results demonstrate that certain derivatives of B₁ and B₆ vitaminsare capable of inhibiting “late” AGE formation. Some of these vitamerssuccessfully inhibited the final levels of AGE produced, in contrast toaminoguanidine, suggesting that they have greater interactions withAmadori or post-Amadori precursors to antigenic AGEs. The Amadori andpost-Amadori intermediates represent a crucial juncture where the“classical” pathway of nonenzymatic glycation begins to becomeessentially irreversible (Scheme I). In earlier inhibition studies“glycation” was usually measured either as Schiff base formed (afterreduction with labeled cyanoborohydride) or as Amadori product formed(after acid precipitation using labeled sugar). Such assays do not yieldinformation on inhibition of post-Amadori conversion steps to “late” AGEproducts, since such steps lead to no change in the amount of labeledsugar that is attached to the proteins. Other “glycation” assays haverelied on the sugar-induced increase of non-specific proteinfluorescence, but this can also be induced by dicarbonyl oxidativefragments of free sugar, such as glycoaldehyde or glyoxal (Hunt et al.,1988, Biochem. 256:205-212), independently of Amadori product formation.

In the case of pyridoxal (PL) and pyridoxal phosphate (PLP), the datasupport the simple mechanism of inhibition involving competitiveSchiff-base condensation of these aldehydes with protein amino groups atglycation sites. Due to internal hemiacetal formation in pyridoxal butnot pyridoxal phosphate, stronger inhibition of post-Amadori AGEformation by PLP is expected by this competitive mechanism. This indeedis observed in the data (FIGS. 1B, 1C, FIGS. 3B, 3C). The inhibition bypyridoxamine is necessarily different, as pyridoxamine lacks an aldehydegroup. However, pyridoxamine is a candidate amine potentially capable offorming a Schiff-base linkage with the carbonyls of open-chain sugars,with dicarbonyl fragments, with Amadori products, or with post-Amadoriintermediates. The mechanism of inhibition of B₁ compounds is notobvious. All the forms contain an amino functionality, so that themarked efficiency of only the pyrophosphate form suggests an importantrequirement for a strong negative charge.

A significant unexpected observation is that the extent of inhibition byaminoguanidine, and some of the other compounds, is considerably less atlate stages of the reaction, than during the early initial phase. Thissuggests a different mechanism of action than that of pyridoxamine andthiamine pyrophosphate, suggesting that the therapeutic potential ofthese compounds will be enhanced by co-administration withaminoguanidine.

EXAMPLE 2 Kinetics of Non-enzymatic Glycation: Paradoxical Inhibition byRibose and Facile Isolation of Protein Intermediate for RapidPost-Amadori AGE Formation

While high concentrations of glucose are used to cause the non-enzymaticglycation of proteins, paradoxically, it was found that ribose at highconcentrations is inhibitory to post-Amadori AGE formation inribonuclease by acting on the post-Amadori “late” stages of theglycation reaction. This unexpectedly inhibitory effect suggests thatthe “early” reactive intermediates, presumably Amadori products, can beaccumulated with little formation of “late” post-Amadori AGE products(AGEs; Maillard products). Investigation into this phenomenon hasdemonstrated: (1) ability to define conditions for the kinetic isolationof Amadori (or post-Amadori) glycated intermediate(s); (2) the abilitystudy the fast kinetics of buildup of such an intermediate; (3) theability to study the surprisingly rapid kinetics of conversion of suchintermediates to AGE products in the absence of free or reversibly boundsugar; (4) the ability to use these intermediates to study andcharacterize inhibition of post-Amadori steps of AGE formation thusproviding a novel system to investigate the mechanism of reaction andthe efficacy of potential agents that could block AGE formation; and (5)with this knowledge it is also further possible to use ¹³C NMR to studythe reactive intermediates and monitor their conversion to variouscandidate AGEs (Khalifah et al., 1996, Biochemistry 35(15):4645-4654).

Chemicals and Materials

As in Example 1 above.

Preparation of Polyclonal Antibodies to AGEs

As in Example 1 above.

ELISA Detection AGE Products

As in Example 1 above.

Amino Acid Analysis

Amino acid analyses were carried out at the Biotechnology SupportFacility of the Kansas University Medical Center. Analyses wereperformed after hydrolysis of glycated protein (reduced with sodiumcyanoborohydride) with 6 N HCl at 110° C. for 18-24 h. Phenylisothiocyanate was used for derivatization, and PTH derivatives wereanalyzed by reverse-phase HPLC on an Applied Biosystems amino acidanalyzer (420A derivatizer, 130A separation system, 920A data analysissystem).

Pentosidine Reverse-phase HPLC Analysis

Pentosidine production in RNase was quantitated by HPLC (Sell & Monnier,1989, J. Biol. Chem. 264:21597-21602; Odetti et al., 1992, Diabetes41:153-159). Ribose-modified protein samples were hydrolyzed in 6 N HClfor 18 h at 100° C. and then dried in a Speed Vac. The samples were thenredissolved, and aliquots were taken into 0.1% trifluoroacetic acid andanalyzed by HPLC on a Shimadzu system using a Vydac C-18 columnequilibrated with 0.1% TFA. A gradient of 0-6% acetonitrile (0.1% inTFA) was run in 30 min at a flow rate of about 1 ml/min. Pentosidine wasdetected by 335 nm excitation/385 nm emission fluorescence, and itselution time was determined by running a synthesized standard. Due tothe extremely small levels of pentosidine expected (Grandhee & Monnier,1991, J. Biol. Chem. 266:11649-11653; Dyer et al., 1991, J. Biol. Chem.266:11654-11660), no attempt was made to quantitate the absoluteconcentrations. Only relative concentrations were determined from peakareas.

Glycation Modifications

Modification with ribose or glucose was generally done at 37° C. in 0.4M phosphate buffer of pH 7.5 containing 0.02% sodium azide. The highbuffer concentration was always used with 0.5 M ribose modifications.The solutions were kept in capped tubes and opened only to remove timedaliquots that were immediately frozen for later carrying out the variousanalyses. “Interrupted glycation” experiments were carried out by firstincubating protein with the ribose at 37° C. for 8 or 24 h, followed byimmediate and extensive dialysis against frequent cold buffer changes at4° C. The samples were then reincubated by quickly warming to 37° C. inthe absence of external ribose. Aliquots were taken and frozen atvarious intervals for later analysis. Due to the low molecular weight ofRNase, protein concentrations were remeasured after dialysis even whenlow molecular weight cut-off dialysis tubing was used. An alternativeprocedure was also devised (see below) in which interruption wasachieved by simple 100-fold dilution from reaction mixtures containing0.5 M ribose. Protein concentrations were estimated from UV spectra. Thedifference in molar extinction between the peak (278 nm) and trough (250nm) was used for RNase concentration determinations in order tocompensate for the general increase in UV absorbance that accompaniesglycation. Time-dependent UV-difference spectral studies were carriedout to characterize the glycation contributions of the UV spectrum.

Data Analysis and Numerical Simulations of Kinetics

Kinetic data were routinely fit to monoexponential or biexponentialfunctions using nonlinear least-squares methods. The kinetic mechanismsof Schemes 5-6 have been examined by numerical simulations of thedifferential equations of the reaction. Both simulations and fitting toobserved kinetics data were carried out with the SCIENTIST 2.0 softwarepackage (Micromath, Inc.). Determination of apparent half-times (FIG.6B) from kinetic data fit to two-exponential functions (FIG. 6A) wascarried out with the “solve” function of MathCAD 4.0 software (MathSoft,Inc.).

RESULTS

Comparison of Glycation by Glucose and Ribose

The reaction of RNase A with ribose and glucose has been followedprimarily with ELISA assays, using R479 rabbit AGE-specific antibodiesdeveloped against glucose-modified BSA. To a lesser extent, theproduction of pentosidine, the only known acid-stable fluorescent AGE,was quantiated by HPLC following acid hydrolysis. Preliminary studiesusing 0.05 M ribose at 37° C. showed that the rate of antigenic AGEformation appears to be modestly increased (roughly 2-3 fold as measuredby the apparent half-time) as the pH is increased from 5.0 to 7.5, withan apparent small induction period at the beginning of the kinetics inall cases. The glycation of RNase with 0.05 M ribose at pH 7.5(half-time near 6.5 days) appears to be almost an order of magnitudefaster than that of glycation with 1.0 M glucose (half-time in excess of30 days; see FIG. 7B, solid line). The latter kinetics also displayed asmall induction period but incomplete leveling off even after 60 days,making it difficult to estimate a true half-time.

When the dependence of the kinetics on ribose concentration was examinedat pH 7.5, a most unexpected result was obtained. The rate of reactioninitially increased with increasing ribose concentration, but atconcentrations above 0.15 M the rate of reaction leveled off and thensignificantly decreased (FIG. 6A). A plot of the dependence of thereciprocal half-time on the concentration of ribose (FIG. 6B) shows thathigh ribose concentrations are paradoxically inhibitory to post-Amadoriantigenic AGE formation. This unusual but consistent effect was found tobe independent of changes in the concentration of either buffer (2-fold)or RNase (10-fold), and it was not changed by affinity purification ofthe R479 antibody on a column of immobilized AGE-RNase. It is also notdue to effects of ribose on the ELISA assay itself. The measuredinhibitory effect by ribose on post-Amadori AGE formation is not likelydue to ribose interference with antibody recognition of the AGEantigenic sites on protein in the ELISA assay. Prior to the firstcontact with the primary anti-AGE antibody on the ELISA plates, glycatedprotein has been diluted over 1000-fold, washed extensively withTween-20 after adsorption, and blocked with a 1% casein coating followedby further washing with Tween-20.

Kinetics of Formation of Post-Amadori Antigenic AGEs by “InterruptedGlycation”

In view of the small induction period seen, an attempt was made todetermine whether there was some accumulation during the reaction, of anearly precursor such as an Amadori intermediate, capable of producingthe ELISA-detectable post-Amadori antigenic AGEs. RNase was glycated atpH 7.5 and 37° C. with a high ribose concentration of 0.5 M, and thereaction was interrupted after 24 h by immediate cooling to 4° C. anddialysis against several changes of cold buffer over a period of 24 h toremove free and reversibly bound (Schiff base) ribose. Such aribose-free sample was then rapidly warmed to 37° C. without re-addingany ribose, and was sampled for post-Amadori AGE formation over severaldays. The AGE antigen production of this 24 h “interrupted glycation”sample is shown by the dashed line and open triangles in FIG. 7A, thetime spent in the cold dialysis is not included. An uninterruptedcontrol (solid line and filled circles) is also shown for comparison.Dramatically different kinetics of post-Amadori antigenic AGE formationare evident in the two samples. The kinetics of AGE antigen productionof the ribose-free interrupted sample now show (1) monoexponentialkinetics with no induction period, (2) a greatly enhanced rate ofantigenic AGE formation, with remarkable half-times of the order of 10h, and (3) production of levels of antigen comparable to those seen inlong incubations in the continued presence of ribose (see FIG. 6A).Equally significant, the data also demonstrate that negligible AGEantigen was formed during the cold dialysis period, as shown by thesmall difference between the open triangle and filled circle points attime 1 day in FIG. 7A. Very little, if any, AGE was formed by the“interruption” procedure itself. These observations show that a fullycompetent isolatable intermediate or precursor to antigenic AGE has beengenerated during the 24 h contact with ribose prior to the removal ofthe free and reversibly bound sugar.

Samples interrupted after only 8 h produced a final amount of AGEantigen that was about 72% of the 24 h interrupted sample. Samples ofRNase glycated with only 0.05 M ribose and interrupted at 8 h by colddialysis and reincubation at 37° C. revealed less than 5% production ofELISA-reactive antigen after 9 days. Interruption at 24 h, however,produced a rapid rise of ELISA antigen (similar to FIG. 7A) to a levelroughly 50% of that produced in the uninterrupted presence of 0.05 Mribose.

The same general interruption effects were also seen with other proteins(BSA and Hemoglobin). Except for a somewhat different absolute value ofthe rate constants, and the amount of antigenic AGEs formed during the24 h 0.5 M ribose incubation, the same dramatic increase in the rate ofAGE antigen formation was observed after removal of 0.5 M ribose.

Glycation is much slower with glucose than with ribose (note thedifference in time scales between FIG. 7A and FIG. 7B). However, unlikethe case with ribose, interruption after 3 days of glycation by 1.0 Mglucose produced negligible buildup of precursor to ELISA-reactive AGEantigens (FIG. 7B, dashed curve).

Kinetics of Pentosidine Formation

The samples subjected to ELISA testing were also assayed for theproduction of pentosidine, an acid-stable AGE. The content ofpentosidine was measured for the same RNase samples analyzed forantibody reactivity by ELISA. Glycation by ribose in 0.4 M phosphatebuffer at pH 7.5 produced pentosidine in RNase A that was quantitated byfluroescence after acid hydrolysis. FIG. 8A shows that underuninterrupted conditions, 0.05 M ribose produces a progressive increasein pentosidine. However, when glycation is carried out under“interrupted” conditions using 0.5 M ribose, a dramatic increase in therate of pentosidine formation is seen immediately after removal ofexcess ribose (FIG. 8B), which is similar to, but slightly more rapidthan, the kinetics of the appearance of antigenic AGEs (FIG. 7A). Agreater amount of pentosidine was also produced with 24 h interruptionas compared with 8 h. Reproducible differences between the kinetics offormation of pentosidine and antigenic AGEs can also be noted. Asignificant amount of pentosidine is formed during the 24 h incubationand also during the cold dialysis, resulting in a jump of the dashedvertical line in FIG. 8B. Our observations thus demonstrate that apentosidine precursor accumulates during ribose glycation that canrapidly produce pentosidine after ribose removal (cf. Odetti et al.,1992, Diabetes 41:153-159).

Rate of Buildup of the Reactive Intermediate(s)

The “interrupted glycation” experiments described above demonstrate thata precursor or precursors to both post-Amadori antigenic AGEs andpentosidine can be accumulated during glycation with ribose. Thekinetics of formation of this intermediate can be independently followedand quantitated by a variation of the experiments described above. Theamount of intermediate generated in RNase at different contact timeswith ribose can be assayed by the maximal extent to which it can produceantigenic AGE after interruption. At variable times after initiatingglycation, the free and reversibly-bound ribose is removed by dialysisin the cold or by rapid dilution (see below). Sufficient time (5 days,which represents several half-lives according to FIG. 7A) is thenallowed after warming to 37° C. for maximal development of post-Amadoriantigenic AGEs. The ELISA readings 5 days after each interruption point,representing maximal AGE development, would then be proportional to theintermediate concentration present at the time of interruption.

FIG. 9 shows such an experiment where the kinetics of intermediatebuildup are measured for RNase A in the presence of 0.5 M ribose (solidsymbols and curve). For comparison, the amount of AGE present beforeribose removal at each interruption point is also shown (open symbolsand dashed lines). As expected (cf. FIG. 7A), little AGE is formed priorto removal (or dilution) of ribose, so that ELISA readings after the 5day secondary incubation periods are mostly a measure of AGE formedafter ribose removal. The results in FIG. 9 show that the rate ofbuildup of intermediate in 0.5 M ribose is exponential and very fast,with a half-time of about 3.3 h. This is about 3-fold more rapid thanthe observed rate of conversion of the intermediate to antigenic AGEsafter interruption (open symbols and dashed curve FIG. 7A).

In these experiments the removal of ribose at each interruption time wasachieved by 100-fold dilution, and not by dialysis. Simple dilutionreduced the concentration of ribose from 0.05 M to 0.005 M. It wasindependently determined (FIG. 6A) that little AGE is produced in thistime scale with the residual 5 mM ribose. This dilution approach wasprimarily dictated by the need for quantitative point-to-point accuracy.Such accuracy would not have been achieved by the dialysis procedurethat would be carried out independently for each sample at eachinterruption point. Our results show that dilution was equivalent todialysis.

A separate control experiment (see FIG. 10 below) demonstrated that theinstantaneous 100-fold dilution gave nearly identical results to thedialysis procedure. These control experiments demonstrate that thereversible ribose-protein binding (Schiff base) equilibrium is quiterapid on this time scale. This is consistent with data of Bunn andHiggins (1981, Science 213: 222-224) that indicated that the half-timeof Schiff base formation with 0.5 M ribose should be on the order of afew minutes. The 100-fold rapid dilution method to reduce ribose is avalid method where quantitative accuracy is essential and cannot beachieved by multiple dialysis of many samples.

Direct Inhibition of Post-Amadori AGE Formation From the Intermediate byRibose and Glucose

The increase in the rate of AGE formation after interruption and sugardilution suggests, but does not prove, that high concentrations ofribose are inhibiting the reaction at or beyond the first “stable”intermediate, presumably the Amadori derivative (boxed in Scheme I). Atest of this was then carried out by studying the effect of directlyadding ribose, on the post-Amadori reaction. RNase was first incubatedfor 24 h in 0.5 M ribose in order to prepare the intermediate. Twoprotocols were then carried out to measure possible inhibition of thepost-Amadori formation of antigenic AGEs by different concentrations ofribose. In the first experiment, the 24 h ribated sample was simplydiluted 100-fold into solutions containing varying final concentrationsof ribose ranging from 0.005 M to 0.505 M (FIG. 10A). The rate andextent of AGE formation are clearly seen to be diminished by increasingribose concentrations. Significantly, up to the highest concentration of0.5 M ribose, the kinetics appear exponential and do not show theinduction period that occurs with uninterrupted glycation (FIGS. 6A and7A) in high ribose concentrations.

A second experiment (FIG. 10B) was also conducted in which the 24 hinterrupted sample was extensively dialyzed in the cold to release freeand reversibly bound ribose as well as any inhibitory products that mayhave formed during the 24 h incubation with ribose. Following this,aliquots were diluted 100-fold into varying concentrations of freshlymade ribose, and the formation of antigenic AGE products was monitoredas above. There results were nearly identical to the experiment of FIG.10A where the dialysis step was omitted. In both cases, the rate andextent of AGE formation were diminished by increasing concentrations ofribose, and the kinetics appeared exponential with no induction period.

The question of whether glucose or other sugars can also inhibit theformation of AGEs from the reactive intermediate obtained by interruptedglycation in 0.5 M ribose was also investigated. The effects of glucoseat concentrations of 1.0-2.0 M were tested (data not shown). Glucose wasclearly not as inhibitory as ribose. When the 24 h ribose interruptedsample was diluted 100-fold into these glucose solutions, the amount ofantigenic AGE formed was diminished by about 30%, but there was littledecrease in the apparent rate constant. Again, the kinetics appearedexponential.

Effect of pH on Post-Amadori Kinetics of AGE Formation

The interrupted glycation method was used to investigate the pHdependence of the post-Amadori kinetics of AGE formation from thereactive intermediate. In these experiments, RNase A was first reactedfor 24 h with 0.5 M ribose at pH 7.5 to generate the reactiveintermediate. The kinetics of the decay of the intermediate to AGEs werethen measured by ELISA. FIG. 11 shows that an extremely wide pH range of5.0-9.5 was achievable when the kinetics were measured by initial rates.A remarkable bell-shaped dependence was observed, showing that thekinetics of antigenic AGEs formation are decreased at both acidic andalkaline pH ranges, with an optimum near pH 8.

A single “pH jump” experiment was also carried out on the pH 5.0 samplestudied above which had the slowest rate of antigenic AGE formation.After 12 days at 37° C. in pH 5.0 buffer, the pH was adjusted quickly to7.5, and antigenic AGE formation was monitored by ELISA. Withinexperimental error, the sample showed identical kinetics (same firstorder rate constant) of AGE formation to interrupted glycation samplesthat had been studied directly at pH 7.5 (FIG. 12). In this experiment,the relative amounts of antigenic AGE could not be directly compared onthe same ELISA plate, but the pH-jumped sample appeared to have formedsubstantial though somehow diminished levels of antigenic AGEs. Theseresults demonstrate that intermediate can be prepared free of AGE andstored at pH 5 for later studies of conversion to AGEs.

Inhibition of Post-Amadori AGE Formation by Aminoguanidine

The efficacy of aminoguanidine was tested by this interrupted glycationmethod, i.e., by testing its effect on post-Amadori formation ofantigenic AGEs after removal of excess and reversibly bound ribose. FIG.20A demonstrates that aminoguanidine has modest effects on blocking theformation of antigenic AGEs in RNase under these conditions, with littleinhibition below 50 mM. Approximately 50% inhibition is achieved only ator above 100-250 mM. Note again that in these experiments, the proteinwas exposed to aminoguanidine only after interruption and removal offree and reversibly bound ribose. Comparable results were also obtainedwith the interrupted glycation of BSA (FIG. 20B).

Amino Acid Analysis of Interrupted Glycation Samples

Amino acid analysis was carried out on RNase after 24 h contact with 0.5M ribose (undialyzed), after extensive dialysis of the 24 h glycatedsample, and after 5 days of incubation of the latter sample at 37° C.These determinations were made after sodium cyanoborohydride reduction,which reduces Schiff base present on lysines or the terminal aminogroup. All three samples, normalized to alanine (12 residues), showedthe same residual lysine content (4.0±0.5 out of the original 10 inRNase). This indicates that after 24 h contact with 0.5 M ribose, mostof the formed Schiff base adducts had been converted to Amadori orsubsequent products. No arginine or histidine residues were lost bymodification.

Discussion

The use of rapidly reacting ribose and the discovery of its reversibleinhibition of post-Amadori steps have permitted the dissection anddetermination of the kinetics of different steps of protein glycation inRNase. Most previous kinetic studies of protein “glycation” haveactually been restricted to the “early” steps of Schiff base formationand subsequent Amadori rearrangement. Some kinetic studies have beencarried out starting with synthesized fructosylamines, i.e. small modelAmadori compounds of glucose (Smith and Thornalley, 1992, Eur. J.Biochem. 210:729-739, and references cited therein), but such studies,with few exceptions, have hitherto not been possible with proteins. Onenotable exception is the demonstration by Monnier (Odetti et al., 1992,supra) that BSA partially glycated with ribose can rapidly producepentosidine after ribose removal. The greater reactivity of ribose hasalso proven a distinct advantage in quantitatively defining the timecourse of AGE formation. It is noted that glucose and ribose are bothcapable of producing similar AGE products, such as pentosidine (Grandhee& Monnier, 1991, supra; Dyer et al. 1991, supra), and some ¹³C NMR modelcompound work has been done with ADP-ribose (Cervantes-Laurean et al.,1993, Biochemistry 32:1528-1534). The present work shows that antigenicAGE products of ribose fully cross-react with anti-AGE antibodiesdirected against glucose-modified proteins, suggesting that ribose andglucose produce similar antigenic AGEs. The primary kinetic differencesobserved between these two sugars are probably due to relativedifferences in the rate constants of steps leading to post-Amadori AGEformation, rather than in the mechanism.

The results presented reveal a marked and paradoxical inhibition ofoverall AGE formation by high concentrations of ribose (FIG. 6) that hasnot been anticipated by earlier studies. This inhibition is rapidlyreversible in the sense that it is removed by dialysis of initiallymodified protein (FIG. 7A) or by simple 100-fold dilution (as used inFIG. 11). The experiments of FIG. 10 demonstrate that it is not due tothe accumulation of dialyzable inhibitory intermediates during theinitial glycation, since dialysis of 24 h modified protein followed byaddition of different concentrations of fresh ribose induces the sameinhibition. The data of FIGS. 10A,B show that the inhibition occurs byreversible and rapid interaction of ribose with protein intermediatecontaining reactive Amadori products. The inhibition is unlikely toapply to the early step of formation of Amadori product due to the rapidrate of formation of the presumed Amadori intermediate that wasdetermined in the experiment of FIG. 9. The identification of thereactive intermediate as an Amadori product is well supported by theamino acid analysis carried out (after sodium cyanoborohydratereduction) before and after dialysis at the 24 h interruption point. Theunchanged residual lysine content indicates that any dischageable Schiffbases have already been irreversibly converted (presumably by Amadorirearrangement) by the 24 h time.

The secondary ribose suppression of “late” but not “early” glycationsteps significantly enhances the accumulation of a fully-competentreactive Amadori intermediate containing little AGE. Its isolation bythe interruption procedure is of importance for kinetic and structuralstudies, since it allows one to make studies in the absence of free orSchiff base bound sugar and their attendant reactions and complications.For example, the post-Amadori conversion rates to antigenic AGE andpentosidine AGE products have been measured (FIG. 7A, open symbols, andFIG. 8B), and demonstrated to be much faster (t½˜10 h) than reflected inthe overall kinetics under uninterrupted conditions (FIG. 6A and FIG.8A). The rapid formation of pentosidine that was measured appearsconsistent with an earlier interrupted ribose experiment on BSA byOdetti et al. (1992, supra). Since ribose and derivatives such asADP-ribose are normal metabolites, the very high rates of AGE formationseen here suggest that they should be considered more seriously assources of potential glycation in various cellular compartments(Cervantes-Laurean et al., 1993, supra), even though theirconcentrations are well below those of the less reactive glucose.

Another new application of the isolation of intermediate is in studyingthe pH dependence of this complex reaction. The unusual bell-shaped pHprofile seen for the post-Amadori AGE formation (FIG. 11) is in strikingcontrast to the mild pH dependence of the overall reaction. The latterkinetics reflect a composite effect of pH on all steps in the reaction,including Schiff base and Amadori product formation, each of which mayhave a unique pH dependence. This complexity is especially wellillustrated by studies of hemoglobin glycation (Lowery et al., 1985, J.Biol. Chem. 260:11611-11618). The bell-shaped pH profile suggests, butdoes not prove, the involvement of two ionizing groups. If true, thedata may imply the participation of a second amino group, such as from aneighboring lysine, in the formation of dominant antigenic AGEs. Theobserved pH profile and the pH-jump observations described suggest thata useful route to isolating and maintaining the reactive intermediatewould be by the rapid lowering of the pH to near 5.0 after 24 hinterruption.

The kinetic studies provide new insights into the mechanisms of actionof aminoguanidine (guanylhydrazine), an AGE inhibitor proposed by Ceramiand co-workers to combine with Amadori intermediates (Brownlee et al.,1986, supra). This proposed pharmacological agent is now in Phase IIIclinical trials for possible therapeutic effects in treating diabetes(Vlassara et al., 1994, supra). However, its mechanism of AGE inhibitionis likely to be quite complex, since it is multifunctional. As anucelophilic hydrazine, it can reversibly add to active carbonyls,including aldehydo carbonyls of open-chain glucose and ribose (Khatamiet al., 1988, Life Sci. 43:1725-1731; Hirsch et al., 1995, Carbohyd.Res. 267:17-25), as well as keto carbonyls of Amadori compounds. It isalso a guanidinium compound that can scavange highly reactive dicarbonylglycation intermediates such as glyoxal and glucosones (Chen & Cerami,1993, J. Carbohyd. Chem. 12:731-742; Hirsch et al., 1992, Carbohyd. Res.232:125-130; Ou & Wolff, 1993, Biochem. Pharmacol. 46:1139-1144). Theinterrupted glycation method allowed examination of aminoguanidineefficacy on only post-Amadori steps of AGE formation. Equally important,it allowed studies in the absence of free sugar or dicarbonyl-reactivefragments from free sugar (Wolff & Dean, 1987, Biochem. J. 245:243-250;Wells-Knecht et al., 1995, Biochemistry 34:3702-3709) that can combinewith aminoguanidine. The results of FIG. 20 demonstrate thataminoguanidine has, at best, only a modest effect on post-Amadori AGEformation reactions, achieving 50% inhibition at concentrations above100-250 mM. The efficacy of aminoguanidine thus predominantly ariseseither from inhibiting early steps of glycation (Schiff base formation)or from scavenging highly reactive dicarbonyls generated duringglycation. Contrary to the original claims, it does not appear toinhibit AGE formation by complexing the Amadori intermediate.

The use of interrupted glycation is not limited for kinetic studies.Interrupted glycation can simplify structural studies of glycatedproteins and identifying unknown AGEs using techniques such as ¹³C NMRthat has been used to detect Amadori adducts of RNase (Neglia et al.,1983, J. Biol. Chem. 258:14279-14283; 1985, J. Biol. Chem.260:5406-5410). The combined use of structural and kinetic approachesshould also be of special interest. For example, although the identityof the dominant antigenic AGEs reacting with the polyclonal antibodiesremains uncertain, candidate AGEs, such as the recently proposed(carboxymethyl)lysine (Reddy et al., 1995, Biochemistry 34:10872-10878;cf. Makita et al., 1992, J. Biol. Chem. 267:5133-5138) should displaythe same kinetics of formation from the reactive intermediate that wehave observed. The availability of the interrupted kinetics approachwill also help to determine the importance of the Amadori pathway to theformation of this AGE. Similarly, monitoring of the interruptedglycation reaction by techniques such as ¹³C NMR should identifyresonances of other candidate antigenic AGEs as being those displayingsimilar kinetics of appearance. Table I lists the ¹³C NMR peaks of theAmadori intermediate of RNase prepared by reaction with C-2 enrichedribose. The downfield peak near 205 ppm is probably due to the carbonylof the Amadori product. In all cases, the ability to remove excess freeand Schiff base sugars through interrupted glycation will considerablysimplify isolation, identification, and structural characterization.

Table I lists the peaks that were assigned to the Post-AmadoriIntermediate due to their invariant or decreasing intensity with time.Peak positions are listed in ppm downfield from TMS.

TABLE I 125 MHz C-13 NMR Resonances of Ribonuclease Amadori IntermediatePrepared by 24 HR Reaction with 99% [2-C13] Ribose 216.5 ppm 108.5 ppm211.7 105.9 208 103.9 103 172 95.8 165 163.9 73.65 162.1 70.2 69.7

Ribonuclease A was reacted for 24 hr with 0.5 M ribose 99% enriched atC-2, following which excess and Schiff base bound ribose was removed byextensive dialysis in the cold. The sample was then warmed back to 37°C. immediately before taking a 2 hr NMR scan. The signals from RNasereacted with natural abundance ribose under identical conditions werethen subtracted from the NMR spectrum. Thus all peaks shown are due toenriched C-13 that originated at the C-2 position. Some of the peaksarise from degradation products of the intermediate, and these can beidentified by the increase in the peak intensity over time. FIG. 31shows the NMR spectrum obtained.

EXAMPLE 3 In Vitro Inhibition of the Formation of Antigenic AdvancedGlycation End-products (AGEs) by Derivatives of Vitamins B₁ and B₆ andAminoguanidine Inhibition of Post-Amadori Kinetics Differs From That ofOverall Glycation

The interrupted glycation method for following post-Amadori kinetics ofAGE formation allows for the rapid quantitative study of “late” stagesof the glycation reaction. Importantly, this method allows forinhibition studies that are free of pathways of AGE formation whicharise from glycoxidative products of free sugar or Schiff base (Namikipathway) as illustrated in Scheme I. Thus the interrupted glycationmethod allows for the rapid and unique identification andcharacterization of inhibitors of “late” stages of glycation which leadto antigenic AGE formation.

Among the vitamin B₁ and B₆ derivatives examined, pyridoxamine andthiamine pyrophosphate are unique inhibitors of the post-Amadori pathwayof AGE formation. Importantly, it was found that efficacy of inhibitionof overall glycation of protein, in the presence of high concentrationsof sugar, is not predictive of the ability to inhibit the post-Amadoristeps of AGE formation where free sugar is removed. Thus whilepyridoxamine, thiamine pyrophosphate and aminoguanidine are potentinhibitors of AGE formation in the overall glycation of protein byglucose, aminoguanidine differs from the other two in that it is not aneffective inhibitor of post-Amadori AGE formation. Aminoguanidinemarkedly slows the initial rate of AGE formation by ribose underuninterrupted conditions, but has no effect on the final levels ofantigenic AGEs produced. Examination of different proteins (RNase, BSAand hemoglobin), confirmed that the inhibition results are generallynon-specific as to the protein used, even though there are individualvariations in the rates of AGE formation and inhibition.

Chemicals and Materials

As in Example 1 above.

Preparation of Polyclonal Antibodies to AGEs

As in Example 1 above.

ELISA Detection of AGE Products

As in Example 1 above.

Uninterrupted Ribose Glycation Assays

Bovine serum albumin, ribonuclease A, and human hemoglobin wereincubated with ribose at 37° C. in 0.4 M sodium phosphate buffer of pH7.5 containing 0.02% sodium azide. The protein (10 mg/ml or 1 mg/ml),0.05 M ribose, and prospective inhibitors (at 0.5, 3, 15 and 50 mM) wereintroduced into the incubation mixture simultaneously. Solutions werekept in the dark in capped tubes. Aliquots were taken and immediatelyfrozen until analyzed by ELISA at the conclusion of the reaction. Theincubations were for 3 weeks (Hb) or 6 weeks (RNase, BSA). Glycationreactions were monitored for constant pH throughout the duration of theexperiments.

Interrupted (Post-Amadori) Ribose Glycation Assays

Glycation was first carried out by incubating protein (10 mg/ml) with0.5 M ribose at 37° C. in 0.4 M phosphate buffer at pH 7.5 containing0.2% sodium azide for 24 h in the absence of inhibitors. Glycation wasthen interrupted to remove excess and reversibly bound (Schiff base)sugar by extensive dialysis against frequent cold buffer changes at 4°C. The glycated intermediate samples containing maximal amount ofAmadori product and little AGE (depending on protein) were then quicklywarmed to 37° C. without re-addition of ribose. This initiatedconversion of Amadori intermediates to AGE products in the absence orpresence of various concentrations (typically 3, 15 and 50 mM) ofprospective inhibitors. Aliquots were taken and frozen at variousintervals for later analysis. The solutions were kept in capped in tubesand opened only to remove timed aliquots that were immediately frozenfor later carrying out the various analyses.

Numerical Analysis of Kinetics Data

Kinetics data (time progress curves) was routinely fit to mono- orbi-exponential functions using non-linear least squares methodsutilizing either SCIENTIST 2.0 (MicroMath, Inc.) or ORIGIN (Microcal,Inc.) software that permit user-defined functions and control ofparameters to iterate on. Standard deviations of the parameters of thefitted functions (initial and final ordinate values and rate constants)were returned as measures of the precision of the fits. Apparenthalf-times for bi-exponential kinetics fits were determined with the“solve” function of MathCad software (MathSoft, Inc.).

RESULTS

Inhibition by Vitamin B₆ Derivatives of the Overall Kinetics of AGEFormation From Ribose.

The inhibitory effects of vitamin B₁ and B₆ derivatives on the kineticsof antigenic AGE formation were evaluated by polyclonal antibodiesspecific for AGEs. Initial inhibition studies were carried out on theglycation of bovine ribonuclease A (RNase) in the continuous presence of0.05 M ribose, which is the concentration of ribose where the rate ofAGE formation is near maximal. FIG. 13 (control curves, filledrectangles) demonstrates that the formation of antigenic AGEs on RNasewhen incubated with 0.05 M ribose is relatively rapid, with a half-timeof approximately 6 days under these conditions. Pyridoxal-5′-phosphate(FIG. 13B) and pyridoxal (FIG. 13C) significantly inhibited the rate ofAGE formation on RNase at concentrations of 50 mM and 15 mM.Surprisingly, pyridoxine, the alcohol form of vitamin B₆, alsomoderately inhibited AGE formation on RNase (FIG. 13D). Of the B₆derivatives examined, pyridoxamine at 50 mM was the best inhibitor ofthe “final” levels of AGE formed on RNase over the 6-week time periodmonitored (FIG. 13A).

Inhibition by Vitamin B₁ Derivatives of the Overall Kinetics of AGEFormation From Ribose.

All of the B₁ vitamers inhibited antigenic AGE formation on RNase athigh concentrations, but the inhibition appeared more complex than forthe B₆ derivatives (FIGS. 14A-C). In the case of thiamine pyrophosphateas the inhibitor (FIG. 14A), both the rate of AGE formation and thefinal levels of AGE produced at the plateau appeared diminished. In thecase of thiamine phosphate as the inhibitor (FIG. 14B), and thiamine(FIG. 14C), there appeared to be little effect on the rate of AGEformation, but a substantial decrease in the final level of AGE formedin the presence of the highest concentration of inhibitor. In general,thiamine pyrophosphate demonstrated greater inhibition than the othertwo compounds, at the lower concentrations examined.

Inhibition by Aminoguanidine of the Overall Kinetics of AGE FormationFrom Ribose

Inhibition of AGE formation by aminoguanidine (FIG. 14D) was distinctlydifferent from that seen in the B₁ and B₆ experiments. Increasingconcentration of aminoguanidine decreased the rate of AGE formation onRNase, but did not reduce the final level of AGE formed. The final levelof AGE formed after the 6-weeks was nearly identical to that of thecontrol for all tested concentrations of aminoguanidine.

Inhibition of the Overall Kinetics of AGE Formation in Serum Albumin andHemoglobin From Ribose

Comparative studies were carried out with BSA and human methemoglobin(Hb) to determine whether the observed inhibition was protein-specific.The different derivatives of vitamin B₆ (FIGS. 15A-D) and vitamin B₁(FIGS. 16A-C) exhibited similar inhibition trends when incubated withBSA as with RNase, pyridoxamine and thiamine pyrophosphate being themost effective inhibitors or each family. Pyridoxine failed to inhibitAGE formation on BSA (FIG. 15D). Pyridoxal phosphate and pyridoxal(FIGS. 15B-C) mostly inhibited the rate of AGE formation, but not thefinal levels of AGE formed. Pyridoxamine (FIG. 15A) exhibited someinhibition at lower concentrations, and at the highest concentrationtested appeared to inhibit the final levels of AGE formed moreeffectively than any of the other B₆ derivatives. In the case of B₁derivatives, the overall extent of inhibition of AGE formation with BSA(FIGS. 16A-C), was less than that observed with RNase (FIGS. 14A-C).Higher concentrations of thiamine and thiamine pyrophosphate inhibitedthe final levels of AGEs formed, without greatly affecting the rate ofAGE formation (FIG. 16C). Aminoguanidine again displayed the sameinhibition effects with BSA as seen with RNase (FIG. 16D), appearing toslow the rate of AGE formation without significantly affecting the finallevels of AGE formed.

The kinetics of AGE formation was also examined using Hb in the presenceof the B₆ and B₁ vitamin derivatives, and aminoguanidine. The apparentabsolute rates of AGE formation were significantly higher with Hb thanwith either RNase or BSA. However, the tested inhibitors showedessentially similar behavior. The effects of the vitamin B₆ derivativesare shown in FIG. 17. Pyridoxamine showed the greatest inhibition atconcentrations of 3 mM and above (FIG. 17A), and was most effective whencompared to pyridoxal phosphate (FIG. 17B), pyridoxal (FIG. 17C), andpyridoxine (FIG. 17D). In the case of the B₁ vitamin derivatives (datanot shown), the inhibitory effects were more similar to the BSAinhibition trends than to RNase. The inhibition was only modest at thehighest concentrations tested (50 mM), being nearly 30-50% for all threeB₁ derivatives. The primary manifestation of inhibition was in thereduction of the final levels of AGE formed.

Inhibition by Vitamin B₆ Derivatives of the Kinetics of Post-AmadoriRibose AGE Formation

Using the interrupted glycation model to assay for inhibition of the“late” post-Amadori AGE formation, kinetics were examined by incubatingisolated Amadori intermediates of either RNase or BSA at 37° C. in theabsence of free or reversibly bound ribose. Ribose sugar that wasinitially used to prepare the intermediates was removed by cold dialysisafter an initial glycation reaction period of 24 h. After AGE formationis allowed to resume, formation of AGE is quite rapid (half-times ofabout 10 h) in the absence of any inhibitors. FIG. 18 shows the effectsof pyridoxamine (FIG. 18A), pyridoxal phosphate (FIG. 18B), andpyridoxal (FIG. 18C) on the post-Amadori kinetics of BSA. Pyridoxine didnot produce any inhibition (data not shown). Similar experiments werecarried out on RNase. Pyridoxamine caused nearly complete inhibition ofAGE formation with RNase at 15 mM and 50 mM (FIG. 18D). Pyridoxal didnot show any significant inhibition at 15 mM (the highest tested), butpyridoxal phosphate showed significant inhibition at 15 mM. Pyridoxalphosphate is known to be able to affinity label the active site of RNase(Raetz and Auld, 1972, Biochemistry 11:2229-2236).

With BSA, unlike RNase, a significant amount of antigenic AGE formedduring the 24 h initial incubation with RNase (25-30%), as evidenced bythe higher ELISA readings after removal of ribose at time zero for FIGS.18A-C. For both BSA and RNase, the inhibition, when seen, appears tomanifest as a decrease in the final levels of AGE formed rather than asa decrease in the rate of formation of AGE.

Inhibition by Vitamin B₁ Derivatives of the Kinetics of Post-AmadoriRibose AGE Formation

Thiamine pyrophosphate inhibited AGE formation more effectively than theother B₁ derivatives with both RNase and BSA (FIG. 19). Thiamine showedno effect, while thiamine phosphate showed some intermediate effect. Aswith the B₆ assays, the post-Amadori inhibition was most apparentlymanifested as a decrease in the final levels of AGE formed.

Effects of Aminoguanidine and N^(α)-Acetyl-L-lysine on the Kinetics ofPost-Amadori Ribose AGE Formation

FIG. 20 shows the results of testing aminoguanidine for inhibition ofpost-Amadori AGE formation kinetics with both BSA and RNase. At 50 mM,inhibition was about 20% in the case of BSA (FIG. 20B), and less than15% with RNase (FIG. 20A). The possibility of inhibition by simpleamino-containing functionalities was also tested usingN^(α)-acetyl-L-lysine (FIG. 21), which contains only a free N^(ε)-aminogroup. N^(α)-acetyl-L-lysine at up to 50 mM failed to exhibit anysignificant inhibition of AGE formation.

Discussion

Numerous studies have demonstrated that aminoguanidine is an apparentlypotent inhibitor of many manifestations of nonenzymatic glycation(Brownlee et al., 1986; Brownlee, 1992, 1994, 1995). The inhibitoryeffects of aminoguanidine on various phenomena that are induced byreducing sugars are widely considered as proof of the involvement ofglycation in many such phenomena. Aminoguanidine has recently enteredinto a second round of Phase III clinical trials for ameliorating thecomplications of diabetes thought to be caused by glycation ofconnective tissue proteins due to high levels of sugar.

Data from the kinetic study of uninterrupted “slow” AGE formation withRNase induced by glucose (Example 1) confirmed that aminoguanidine is aneffective inhibitor, and further identified a number of derivatives ofvitamins B₁ and B₆ as equally or slightly more effective inhibitors.However, the inhibition by aminoguanidine unexpectedly appeared todiminish in effect at the later stages of the AGE formation reaction.Due to the slowness of the glycation of protein with glucose, thissurprising observation could not be fully examined. Furthermore, it hasbeen suggested that there may be questions about the long-term stabilityof aminoguanidine (Ou and Wolff, 1993, supra).

Analysis using the much more rapid glycation by ribose allowed for theentire time-course of AGE formation to be completely observed andquantitated during uninterrupted glycation of protein. The use ofinterrupted glycation uniquely allowed further isolation and examinationof only post-Amadori antigenic AGE formation in the absence of free andreversibly bound (Schiff base) ribose. Comparison of the data from thesetwo approaches with the earlier glucose glycation kinetics has providednovel insights into the mechanisms and effectiveness of variousinhibitors. FIG. 22 are bar graphs which depict summarized comparativedata of percent inhibition at defined time points using variousconcentrations of inhibitor. FIG. 22A graphs the data for inhibitionafter interrupted glycation of RNase AGE formation in ribose. FIG. 22Bgraphs the data for inhibition after interrupted glycation of BSA AGEformation by ribose.

The overall results unambiguously demonstrate that aminoguanidine slowsthe rate of antigenic AGE formation in the presence of sugar but haslittle effect on the final amount of post-Amadori AGE formed. Thusobservations limited to only the initial “early” stages of AGE formationwhich indicate efficacy as an inhibitor may in fact be misleading as tothe true efficacy of inhibition of post-Amadori AGE formation. Thus theability to observe a full-course of reaction using ribose andinterrupted glycation gives a more complete picture of the efficacy ofinhibition of post-Amadori AGE formation.

EXAMPLE 4 Animal Model & Testing of In Vivo Effects of AGEFormation/Inhibitors

Hyperglycemia can be rapidly induced (within one or two days) in rats byadministration of streptozocin (aka. streptozotocin, STZ) or alloxan.This has become a common model for diabetes melitus. However, these ratsmanifest nephropathy only after many months of hyperglycemia, andusually just prior to death from end-stage renal disease (ESRD). It isbelieved that this pathology is caused by the irreversible glucosechemical modification of long-lived proteins such as collagen of thebasement membrane. STZ-diabetic rats show albuminuria very late afterinduction of hyperglycemia, at about 40 weeks usually only just prior todeath.

Because of the dramatic rapid effects of ribose demonstrated in vitro inthe examples above, it was undertaken to examine the effects of riboseadministration to rats, and the possible induction of AGEs by the rapidribose glycation. From this study, a rat model for accelerated riboseinduced pathology has been developed.

Effects of Very Short-term Ribose Administration In Vivo

Phase I Protocol

Two groups of six rats each were given in one day either:

a. 300 mM ribose (two intraperitoneal infusions 6-8 hours apart, each 5%of body weight as ml); or

b. 50 mM ribose (one infusion)

Rats were then kept for 4 days with no further ribose administration, atwhich time they were sacrificed and the following physiologicalmeasurements were determined: (i) initial and final body weight; (ii)final stage kidney weight; (iii) initial and final tail-cuff bloodpressure; (iv) creatinine clearance per 100 g body weight.

Albumin filtration rates were not measured, since no rapid changes wereinitially anticipated. Past experience with STZ-diabetic rats shows thatalbuminuria develops very late (perhaps 40 weeks) after the induction ofhyperglycemia and just before animals expire.

Renal Physiology Results

a. Final body weight and final single kidney weight was same for low andhigh ribose treatment groups.

b. Tail-cuff blood pressure increased from 66±4 to 83±3 to rats treatedwith low ribose (1×50 mM), and from 66±4 to 106±5 for rats treated withhigh ribose (2×300 mM). These results are shown in the bar graph of FIG.23.

c. Creatinine clearance, as cc per 100 g body weight, was decreased(normal range expected about 1.0-1.2) in a dose-dependent fashion to0.87±0.15 for the low ribose group, and decreased still further 30% to0.62±0.13 for the high ribose group. These results are shown in the bargraph of FIG. 24.

Phase I Conclusion

A single day's ribose treatment caused a dose-dependent hypertension anda dose-dependent decrease in glomerular clearance function manifest 4days later. These are significant metabolic changes of diabetes seenonly much later in STZ-diabetic rats. These phenomenon can behypothesized to be due to ribose irreversible chemical modification(glycation) of protein in vivo.

Effect of Exposure to Higher Ribose Concentrations for Longer Time

Phase II Protocol

Groups of rats (3-6) were intraperitoneally given 0.3 M “low ribosedose” (LR) or 1.0 M “high ribose dose” (HR) by twice-daily injectionsfor either (i) 1 day, (ii) a “short-term” (S) of 4 days, or (iii) a“long-term” (L) of 8 days. Additionally, these concentrations of ribosewere included in drinking water.

Renal Physiology Results

a. Tail-cuff blood pressure increased in all groups of ribose-treatedrats, confirming Phase I results. (FIG. 23).

b. Creatinine clearance decreased in all groups in a ribosedose-dependent and time-dependent manner (FIG. 24).

c. Albumin Effusion Rate (AER) increased significantly in aribose-dependent manner at 1-day and 4-day exposures. However, it showedsome recovery at 8 day relative to 4 day in the high-dose group but notin the low-dose group. These results are shown in the bar graph of FIG.25.

d. Creatinine clearance per 100 g body weight decreased for both low-and high-ribose groups to about the same extent in a time-dependentmanner (FIG. 24).

Phase II Conclusion

Exposure to ribose for as little as 4 days leads to hypertension andrenal dysfunction, as manifest by both decreased creatinine clearanceand increased albumin filtration. These changes are typical of diabetesand are seen at much later times in STZ-diabetic rats.

Intervention by Two New Therapeutic Compounds and Aminoguanidine

Phase III Protocol

Sixty rats were randomized into 9 different groups, including thoseexposed to 1 M ribose for 8 days in the presence and absence ofaminoguanidine, pyridoxamine, and thiamine pyrophosphate as follows:

Control Groups:

(i) no treatment;

(ii) high dose (250 mg/kg body weight) of pyridoxamine (“compound-P”);

(iii) high dose (250 mg/kg body weight of thiamine pyrophosphate(“compound-T” or “TPP”); and

(iv) low dose (25 mg/kg body weight) of aminoguanidine (“AG”).

Test Groups:

(i) only 1 M ribose-saline (2×9 cc daily IP for 8 days);

(ii) ribose plus low dose (“LP”) of pyridoxamine (25 mg/kg body weightinjected as 0.5 ml with 9 cc ribose);

(iii) ribose plus high dose (“HP”) of pyridoxamine (250 mg/kg bodyweight injected as 0.5 ml with 9 cc ribose);

(iv) ribose plus high dose (“HT”) of thiamine pyrophosphate (250 mg/kgbody weight injected as 0.5 ml with 9 cc ribose); and

(v) ribose plus low dose of amino guanidine (25 mg/kg body weightinjected as 0.5 ml with 9 cc ribose).

Unlike Phase II, no ribose was administered in drinking water.Intervention compounds were pre-administered for one day prior tointroducing them with ribose.

Renal Physiology Results

a. Blood pressure was very slightly increased by the three compoundsalone (control group); ribose-elevated BP was not ameliorated by theco-administration of compounds. These results are shown in the bar graphof FIG. 26.

b. Creatinine clearance in controls was unchanged, except for TPP whichdiminished it.

c. Creatinine clearance was normalized when ribose was co-administeredwith low dose (25 mg/kg) of either aminoguanidine or pyridoxamine. Theseresults are shown in the bar graph of FIG. 27.

d. High concentrations (250 mg/kg) or pyridoxamine and TPP showed onlypartial protection against the ribose-induced decrease in creatinineclearance (FIG. 27).

e. Albumin effusion rate (AER) was elevated by ribose, as well as byhigh dose of pyridoxamine and TPP, and low dose of aminoguanidine in theabsence of ribose. These results are shown in the bar graph of FIG. 28.

f. Albumin effusion rate was restored to normal by the co-administrationof low dose of both aminoguanidine and pyridoxamine. These results areshown in the bar graph of FIG. 29.

Phase III Conclusions

As measured by two indicies of renal function, pyridoxamine andaminoguanidine, both at 25 mg/kg, were apparently effective, and equallyso, in preventing the ribose-induced decrease in creatinine clearanceand ribose-induced mild increase in albuminuria.

(i) Thiamine pyrophosphate was not tested at 25 mg/kg; (ii) thiaminepyrophosphate and pyridoxamine at 250 mg/kg were partially effective inpreventing creatinine clearance decreases but possibly not in preventingmild proteinuria; (iii) at these very high concentrations and in theabsence of ribose, thiamine pyrophosphate alone produced a decrease increatinine clearance, and both produced mild increases in albuminuria.

Summary

Renal Function and Diabetes

Persistent hyperglycemia in diabetes mellitus leads to diabeticnephropathy in perhaps one third of human patients. Clinically, diabeticnephropathy is defined by the presence of:

1. decrease in renal function (impaired glomerular clearance)

2. an increase in urinary protein (impaired filtration)

3. the simultaneous presence of hypertension

Renal function depends on blood flow (not measured) and the glomerularclearance, which can be measured by either inulin clearance (notmeasured) or creatinine clearance. Glomerular permeability is measuredby albumin filtration rate, but this parameter is quite variable. It isalso a log-distribution function: a hundred-fold increase in albuminexcretion represents only a two-fold decrease in filtration capacity.

Ribose Diabetic Rat Model

By the above criteria, ribose appears to very rapidly inducemanifestations of diabetic nephropathy, as reflected in hypertension,creatinine clearance and albuminuria, even though the latter is notlarge. In the established STZ diabetic rat, hyperglycemia is rapidlyestablished in 1-2 days, but clinical manifestations of diabeticnephropathy arise very late, perhaps as much as 40 weeks foralbuminuria. In general, albuminuria is highly variable from day to dayand from animal to animal, although unlike humans, most STZ rats doeventually develop nephropathy.

Intervention by Compounds

Using the ribose-treated animals, pyridoxamine at 25 mg/kg body weightappears to effectively prevent two of the three manifestations usuallyattributed to diabetes, namely the impairment of creatinine clearanceand albumin filtration. It did so as effectively as aminoguanidine. Theeffectiveness of thiamine pyrophosphate was not manifest, but it shouldbe emphasized that this may be due to its use at elevated concentrationsof 250 mg/kg body weight. Pyridoxamine would have appeared much lesseffective if only the results at 250 mg/kg body weight are considered.

Effect of Compounds Alone

Overall, the rats appeared to tolerate the compounds well. Kidneyweights were not remarkable and little hypertension developed. Thephysiological effects of the compounds were only tested at 250 mg/kg.Thiamine pyrophosphate, but not pyridoxamine, appeared to decreasecreatinine clearance at this concentration. Both appeared to slightlyincrease albuminuria, but these measurements were perhaps the leastreliable.

Human Administration

A typical adult human being of average size weighs between 66-77 Kg.Typically, diabetic patients may tend to be overweight and can be over112 Kg. The Recommended dietary allowances for an adult male of between66-77 Kg, as revised in 1989, called for 1.5 mg per day of thiamine, and2.0 mg per day of Vitamin B₆ (Merck Manual of Diagnosis and Therapy,16th edition (Merck & Co., Rathaway, N.J., 1992) pp 938-939).

Based upon the rat model approach, a range of doses for administrationof pyridoxamine or thiamine pyrophosphate that is predicted to beeffective for inhibiting post-Amadori AGE formation and thus inhibitingrelated pathologies would fall in the range of 1 mg/100 g body weight to200 mg/100 g body weight. The appropriate range when co-administeredwith aminoguanidine will be similar. Calculated for an average adult of75 Kg, the range (at 10 mg/1 Kg body weight) can be approximately 750 mgto upwards of 150 g (at 2 g/1 Kg body weight). This will naturally varyaccording to the particular patient.

EXAMPLE 5 In Vivo Inhibition of the Formation of Advanced GlycationEnd-products (AGES) by Derivatives of Vitamins B₁ and B₆ andAminoguanidine. Inhibition of Diabetic Nephropathy

The interrupted glycation method, as described in the examples above,allows for the rapid generation of stable well-defined protein Amadoriintermediates from ribose and other pentose sugars for use in in vivostudies.

The effects of 25 mg/kg/day pyridoxamine (PM) and aminoguanidine (AG) onrenal pathology induced by injecting Sprague-Dawley rats daily with 50mg/kg/day of ribose-glycated Amadori-rat serum albumin (RSA), AGE-RSA,and unmodified RSA for 6 weeks. Hyperfiltration (increased creatinineclearance) was transiently seen with rats receiving Amadori-RSA andAGE-RSA, regardless of the presence of PM and AG.

Individuals from each group receiving Amadori-RSA and AGE-RSA exhibitedmicroalbuminuria, but none was seen if PM was co-administered.Immunostaining with anti-RSA revealed glomerular staining in ratstreated with AGE-RSA and with Amadori-RSA; and this staining wasdecreased by treatment with PM but not by AG treatment. A decrease inglomerular sulfated glycosaminoglycans (Alcian blue pH 1.0 stain) wasalso found in rats treated with glycated (Amadori and AGE) RSA. Thisappears to be due to reduced heparan sulfate proteoglycans (HSPG), asevidenced by diminished staining with mAb JM-403 that is specific forHSPG side-chain. These HSPG changes were ameliorated by treatment withPM, but not by AG treatment.

Thus we conclude that pyridoxamine can prevent both diabetic-likeglomerular loss of heparan sulfate and glomerular deposition of glycatedalbumin in SD rats chronically treated with ribose-glycated albumin.

Materials and Methods

Chemicals

Rat serum albumin (RSA) (fraction V, essentially fatty acid-free 0.005%;A2018), D-ribose, pyridoxamine, and goat alkaline phosphatase-conjugatedanti-rabbit IgG were all from Sigma Chemicals. Aminoguanidinehydrochloride was purchased from Aldrich Chemicals.

Preparation of Ribated RSA

Rat serum albumin was passed down an Affi-Gel Blue column (Bio-Rad), aheparin-Sepharose CL-6B column (Pharmacia) and an endotoxin-bindingaffinity column (Detoxigel, Pierce Scientific) to remove any possiblecontaminants. The purified rat serum albumin (RSA) was then dialyzed in0.2 M phosphate buffer (pH 7.5). A portion of the RSA (20 mg/ml) wasthen incubated with 0.5 M ribose for 12 hours at 37° C. in the dark.After the 12 hour incubation the reaction mixture was dialyzed in cold0.2 M sodium phosphate buffer over a 36 hour period at 4° C. (thisdialysis removes not only the free ribose, but also the Schiff-baseintermediaries). At this stage of the glycation process, the ribatedprotein is classified as Amadori-RSA and is negative for antigenic AGEs,as determined by antibodies reactive with AGE protein (as describedpreviously;

R618, antigen:glucose modified AGE-Rnase). The ribated protein is thendivided into portions that will be injected either as: a) Amadori-RSA,b) NaBH₄-reduced Amadori-RSA, c) AGE-RSA.

The ribated protein to be injected as Amadori-RSA is simply dialyzedagainst cold PBS at 4° C. for 24 hours. A portion of the Amadori-RSA in0.2 M sodium phosphate is reduced with NaBH₄ to form NaBH₄-reducedAmadori-RSA. Briefly, aliquots were reduced by adding 5 uL of NaBH₄stock solution (100 mg/ml in 0.1 M NaOH) per mg of protein, incubatedfor 1 hour at 37° C., treated with HCl to discharge excess NaBH₄, andthen dialyzed extensively in cold PBS at 4° C. for 36 hours. The AGE-RSAwas formed by reincubating the Amadori-RSA in the absence of sugar for 3days. The mixture was then dialyzed against cold PBS at 4° C. for 24hours. All solutions were filtered (22 um filter) sterilized andmonitored for endotoxins by a limulus amoebocyte lysate assay (E-Toxate,Sigma Chemical) and contained <0.2 ng/ml before being frozen (−70° C.)down into individual aliquots until it was time for injection.

Animal Studies

Male Sprague-Dawley rats (Sasco, 100 g) were used. After a 1 weekadaptation period, rats were placed in metabolic cages to obtain a 24hour urine specimen for 2 days before administration of injections. Ratswere then divided into experimental and control groups and given tailvein injections with either saline, unmodified RSA (50 mg/kg),Amadori-RSA (50 mg/kg), NaBH₄-reduced Amadori-RSA (50 mg/kg), or AGE-RSA(50 mg/kg).

Rats injected with Amadori-RSA and AGE-RSA were then either leftuntreated, or futher treated by the administration of eitheraminoguanidine (AG; 25 mg/kg), pyridoxamine (PM; 25 mg/kg), or acombination of AG and PM (10 mg/kg each) through the drinking water.Body weight and water intake of the rats were monitored weekly in orderto adjust dosages. At the conclusion of the experimental study the ratswere placed in metabolic cages to obtain 24 hour urine specimen for 2days prior to sacrificing the animals.

Total protein in the urine samples was determined by Bio-Rad assay.Albumin in urine was determined by competitive ELISA using rabbitanti-rat serum albumin (Cappell) as primary antibody (1/2000) and goatanti-rabbit IgG (Sigma Chemical) as a secondary antibody (1/2000). Urinewas tested with Multistix 8 SG (Miles Laboratories) for glucose, ketone,specific gravity, blook, pH, protein, nitrite, and leukocytes. Nothingremarkable was detected other than some protein.

Creatinine measurements were performed with a Beckman creatinineanalyzer II. Blood samples were collected by heart puncture beforetermination and were used in the determination of creatinine clearance,blood glucose (glucose oxidase, Sigma chemical), fructosamine (nitrobluetetrazolium, Sigma chemical), and glycated Hb (columns, Piercechemicals). Aorta, heart, both kidneys and the rat tail were visuallyinspected and then quickley removed after perfusing with saline throughthe right ventricle of the heart. One kidney, aorta, rat tail, and thelower ⅔ of the heart were snap-frozen and then permanently stored at−70° C. The other kidney was sectioned by removing both ends (cortex) tobe snap-frozen, with the remaining portions of the kidney beingsectioned into thirds with two portions being placed into neutralbuffered formalin and the remaining third minced and placed in 2.5%glutaraldehyde/2% paraformaldehyde.

Light Microscopy

After perfusion with saline, kidney sections were fixed in ice-cold 10%neutral buffered formalin. Paraffin-embedded tissue sections from allrat groups (n=4 per group) were processed for staining with Harris' alumhematoxylin and eosin (H&E), perodic acid/Schiff reagent (PAS), andalcian blue (pH 1.0 and pH 2.5) stains for histological examination. Thealcian blue sections were scored by two investigators in a blindedfashion.

Electron Microscopy

Tissues were fixed in 2.5% glutaraldehyde/2% paraformaldehyde (0.1 Msodium cacodylate, pH 7.4), post-fixed for 1 hour in buffered osmiumtetroxide (1.0%), prestained in 0.5% uranyl acetate for 1 hour andembedded in Effapoxy resin. Ultrathin sections were examined by electronmicroscopy.

Immunofluorescence

Parrafin-embedded sections were deparaffinized and then blocked with 10%goat serum in PBS for 30 min at room temperature. The sections were thenincubated for 2 hour at 37° C. with primary antibody, either affinitypurified polyclonal rabbit anti-AGE antibody, or a polyclonal sheepanti-rat serum albumin antibody (Cappell). The sections were then rinsedfor 30 min with PBS and incubated with secondary antibody, eitheraffinity purified FITC-goat anti-rabbit IgG (H+L) double stain grade(Zymed) or a Rhodamine-rabbit anti-sheep IgG (whole) (Cappell) for 1hour at 37° C. The sections were then rinsed for 30 min with PBS in thedark, mounted in aqueous mounting media for immunocytochemistry(Biomeda), and cover slipped. Sections were scored in a blinded fashion.Kidney sections were evaluated by the number and intensity of glomerularstaining in 5 regions around the periphery of the kidney. Scores werenormalized for the immunofluorescent score per 100 glomeruli with ascoring system of 0-3.

Preparation of Polyclonal Antibodies to AGE-proteins

Immunogen was prepared by glycation of BSA (R479 antibodies) or Rnase(R618 antibodies) at 1.6 g protein in 15 ml for 60-90 days using 1.5 Mglucose in 0.4 M phosphate containing 0.05% sodium azide at pH 7.4 and37° C. New Zealand white rabbit males of 8-12 weeks were immunized bysubcutaneous administration of a 1 ml solution containing 1 mg/ml ofglycated protein in Freund's adjuvant. The primary injection used thecomplete adjuvant and three boosters were made at three week intervalswith Freund's incomplete adjuvant. The rabbits were bled three weeksafter the last booster. The serum was collected by centrifugation ofclotted whole blood. The antibodies are AGE-specific, being unreactivewith either native proteins or with Amadori intermediates.

ELISA Detection of AGE Products

The general method of Engvall (21) was used to perform the ELISA.Glycated protein samples were diluted to approximately 1.5 ug/ml in 0.1M sodium carbonate buffer of pH 9.5 to 9.7. The protein was coatedovernight at room temperature onto a 96-well polystyrene plate bypippetting 200 ul of protein solution into each well (about 0.3ug/well). After coating, the excess protein was washed from the wellswith a saline solution containing 0.05% Tween-20. The wells were thenblocked with 200 ul of 1% casein in carbonate buffer for 2 hours at 37°C. followed by washing. Rabbit anti-AGE antibodies were diluted at atiter of 1:350 in incubation buffer and incubated for 1 hour at 37° C.,followed by washing. In order to minimize background readings, antibodyR618 used to detect glycated RSA was generated by immunization againstglycated Rnase. An alkaline phosphatase-conjugated antibody to rabbitIgG was then added as the secondary antibody at a titer of 1:2000 andincubated for 1 hour at 37° C., followed by washing. Thep-nitrophenolate being monitored at 410 nm with a Dynatech MR4000microplate reader.

Results

The rats in this study survived the treatments and showed no outwardsigns of any gross pathology. Some of the rats showed some small weightchanges and tail scabbing.

Initial screening of kidney sections with PAS and H&E stains did notreveal any obvious changes, and some EM sections did not reveal anygross changes in the glomerular basement membrane (GBM). However, uponAlcian Blue staining, striking differences were discovered. Alcian bluestaining is directed towards negatively charged groups in tissues andcan be made selective via changes in the pH of staining. At pH 1.0Alcian blue is selective for mucopolysaccharides, and at pH 2.5 detectsglucoronic groups. Thus negative charges are detected depending upon thepH of the stain.

At pH 2.5 Alcian blue staining showed that Amadori-RSA (p<0.05) andAGE-RSA (p<0.01) induced increased staining for acidicglycosaminoglycans (GAG) over control levels (FIG. 33). For both AGE-RSAand Amadori-RSA, treatment with pyridoxamine (PM) prevented the increasein staining (p<0.05 as compared with controls). In contrast, treatmentwith aminoguanidine (AG) or combined PM and AG at 10 mg/kg each, did notprevent the increase.

At pH 1.0 Alcian blue staining was significantly decreased by AGE-RSA(p<0.001) (FIG. 34). However, no significant difference was seen withAmadori-RSA. Due to faint staining, treatment with PM, AG and combinedcould not be quantitated.

Immunofluorescent glomerular staining for RSA showed elevated stainingwith Amadori-RSA and AGE-RSA injected animals (FIG. 35). Significantreduction of this effect was seen in the rats treated with PM, and notwith AG or combined AG & PM.

Immunofluorescent glomerular staining for Heparan Sulfate ProteoglycanCore protein showed slightly reduced staining with Amadori-RSA andAGE-RSA injected animals but were not statistically significant (FIG.36). A reduction of this effect was seen in the rats treated with PM,and not with AG or combined AG & PM. However, immunofluorescentglomerular staining for Heparan Sulfate Proteoglycan side-chain showedhighly reduced staining with Amadori-RSA and AGE-RSA injected animals(FIG. 37) A significant reduction of this effect was seen in the ratstreated with PM, and not with AG or combined AG & PM.

Analysis of average glomerular volume by blinded scoring showed thatAmadori-RSA and AGE-RSA caused significant increase in average glomerulivolume (FIG. 38). A significant reduction of this effect was seen withtreatment of the rats with PM. No effect was seen with treatment with AGor combined AG and PM at 10 mg/kg each.

EXAMPLE 6 Identification of PM-adducts Formed During Oxidation ofLinoleate

Introduction

Modification of the lysine residues of low density lipoprotein (LDL) isthought to be an initiating event in the development of atherosclerosis.Analysis of copper-oxidized LDL (NaBH₄ treated protein) leads tomodification of approximately 32% of the lysine residues. Analysis ofnon-reduced oxidized LDL shows that approximately one-half of themodifications are labile to conditions of acid hydrolysis (Steinbrecher,1987). The majority of the lysine modifications present on oxidized LDLhave not been identified. The only characterized and chemicallymeasurable lipoxidation products are N^(ε)-(carboxymethyl)lysine (CML),N⁶⁸ -(carboxyethyl)lysine (CEL), MDA-lysine and HNE-lysine. Theseproducts, however, account for less than 1% of the reducible lysinemodifications formed during LDL oxidation. To better understand thechanges that occur to LDL in vivo, other, more prevalent lipoxidationproducts need to be identified.

Information on the nature of lysine modifications formed duringmodification of proteins by lipid peroxidation reactions is scant. Partof the difficulty in identifying modifications is the extreme complexityof poly-unsaturated fatty acid (PUFA) oxidation reactions. Not only doPUFAs produce a large number of oxidation products (Spiteller, 1998),but the kinetics of PUFA oxidation and the quantities of oxidationproducts also varies from lab to lab depending on such factors as bufferpH, contaminating metal ions, and temperature. The variations affectingthe rate of PUFA oxidation and the types of products produced becomeeven more complicated upon introduction of protein into the system. Forthis reason, identification of specific and measurable lysinemodifications has been an ongoing challenge.

In vitro copper-catalyzed oxidation of LDL is a commonly used model forstudying covalent modifications that occur to the lysine residues duringoxidation of apoB-100 in vivo. Several well-known products of lipidperoxidation have been used to modify LDL in vitro.Malondialdehyde-modified LDL is taken up by macrophages in vitro ifgreater than 15% of the lysine residues are modified (Haberland, 1982).The α,β-unsaturated aldehyde 4-HNE has been shown to modify LDL morerapidly than MDA (Jürgens, 1986; Uchida, 1994). We have quantified bothMDA- and HNE-adducts to lysine in copper-oxidized LDL (Requena, 1997).Though readily detectable, both products together account for less than1% of the total lysine modifications in copper-oxidized LDL. We hve alsoquantified CML and CEL in oxidized LDL (˜2 and 0.2 mmoles/mole lysine,respectively) but like MDA- and HNE-lysine, these are also present atlow levels, (Fu, 1996).

Our initial goal was to trap products of lipid peroxidation usingpyridoxamine (PM), which has a nucleophilic primary amino group andcould therefore serve as a model for the types of modifications thatmight occur to lysine residues in proteins. PM was easily monitoredbecause of its fluorescence and absorbance properties, and thereforeserved as a convenient chemical “tag” with which new product formationcould be detected. PM was reacted with linoleate (the primary PUFA inLDL) in phosphate buffer at a ratio of 1:5. The reactions were analyzedby RP-HPLC for new product formation. New products, representingPM-adducts to oxidized linoleate, were then isolated and characterizedusing a variety of techniques. We have identified 2 major products, andpropose that these same modifications may account for a significantnumber of the unidentified lysine modifications in vivo.

EXPERIMENTAL PLAN

First, we sought to characterize the types of lysine modifications thatoccur during LDL oxidation in vivo, using model reactions ofpyridoxamine (PM) with linoleic acid (the major PUFA in LDL). Wedescribe experiments showing that the primary amino group of PMcovalently traps products of linoleate oxidation, and that the two majorproducts formed are amide derivatives of PM.

Secondly, based on observations discussed above, we hypothesized that PMwould inhibit the modification of proteins by lipid peroxidationreactions by acting as a “sacrificial nucleophile” or carbonyl trap. Wedisclose experiments in which PM was shown to inhibit lipoxidativemodification in both a model protein system (reaction of bovinepancreatic ribonuclease with arachidonic acid) and duringcopper-catalyzed oxidation of LDL. We also report that PM demonstratedminimal anti-oxidant activity, consistent with its proposed primary rolein trapping reactive carbonyl intermediates formed during lipidperoxidation reactions. This finding is based on measurements of thekinetics of oxidation of linoleate and formation of thiobarbituric acidreactive substances (TBARs) in the presence and absence of PM.

The results demonstrated two important findings. First, the formation ofPM-amide derivatives during reaction of PM with linoleate can be used asmodel for the types of lysine modifications that occur when LDL isoxidized in vivo. This finding is important considering that to thisdate, fewer than 1% of the lysine modifications of oxidized LDL havebeen described. Secondly, the inhibition of lipoxidation reactions by PMsuggests that it may be effective as a therapeutic agent for inhibitingthe development of atherosclerosis and diabetic vascular disease.

Materials

Arachidonic acid, linoleic acid, linoleic acid methyl ester, oleic acid,pyridoxamine (dihydrochloride), pyridoxal (hydrochloride), pyridoxine(hydrochloride), ribonuclease A (Type II-A from bovine pancreas),diethylenetriaminepentaacetic acid, phytic acid, sodium borohydride,borontrichloride-methanol, hexanoyl (caproyl) chloride andaminoguanidine (hemisulfate) were purchased from Sigma.Heptafluorobutyric acid, trifluoracetic acid, acetyl chloride andtrifluoroacetic anhydride were purchased from Acros. Azelaic(nonanedioic) acid monomethyl ester was purchased from Aldrich.Low-density lipoprotein was a gift from Dr. Alicia Jenkins of theDepartment of Medicine, Division of Endocrinology, Medical University ofSouth Carolina.

Methods

Analytical Instrumentation

RP-HPLC was performed on a Waters (Waters Associates, Milford, Mass.)600S controller, 660 HPLC pump equipped with a Waters WISP model 723autosampler, photodiode array detector (model 996) and Shimadzu (Kyoto,Japan) fluorescence detector (RF-535 fluorescence HPLC monitor).

Amino acid analysis was performed using a SSI (Scientific Systems Inc.,State College, Pa.) model 22D HPLC pump and model 232D gradientcontroller, SSI model 50 column oven (maintained at 55° C.) equippedwith a Waters Ultra-WISP model 715 autosampler and Waters model 420-ACfluorescence detector. Amino acids were separated using a sulfonateddivinylbenzene cation-exchange column (3×250 mm) (Pickering Labs,Mountain View, Calif.) and a Pickering buffering system. The buffersused were of the following composition: buffer A (Na315), pH 3.15, 98%water, 2% sodium citrate, 0.6% HCl; buffer B (Na740) pH 7.5, 93% water,5% sodium chloride, 1.4% sodium acetate; buffer C (Na Reagent), pH 13,94% water, 0.4% NaCl, 0.6% gradient used is shown in Table 2.1. Aminoacids were derivatized post-column using o-phthaldialdehyde (OPA) anddetected by fluorescence (Ex=365 nm and Em=425 nm).

TABLE 2.1 TIME % A % B % C FLOW Initial 100 0 0 0.3 10 100 0 0 0.3 40 4060 0 0.3 60 0 100 0 0.3 84 0 100 0 0.3 84.1 0 0 100 0.3 86 0 0 100 0.386.1 100 0 0 0.3 115 100 0 0 0.3

Gas chromatography/mass spectrometry was performed on a Hewlett-Packard(Palo Alto, Calif.) model 5890 gas chromatograph/5970 mass selectivedetector equipped with a Hewlett-Packard model 7673A autosampler. A 30meter HP-5MS (5% phenyl methyl siloxane) capillary column (Restek,Bellefonte, Pa.) was used for GC seperation ESI-MS and LC-ESI-MS wereperformed on a VG-70 triple quadropole mass spectrometer (Analytica ionsource) interfaced to a Hewlett Packard series 1100 HPLC pump anddegasser. Absorbance was monitored with an ISCO (Lincoln, Nebr.) μLC-10absorbance detector. Separations were done on an ODS Aquasil column(Keystone Scientific, Bellefonte, Pa.) using a solvent system of 0.1%HFBA (Buffer A) and acetonitrile (Buffer B). Gradient conditions areshown in Table 2.2.

TABLE 2.2 TIME % A % B FLOW (ml/min) Initial 100 0 0.05 2 100 0 0.05 840 60 0.05 12 40 60 0.05 15 25 75 0.05 25 25 75 0.05

Spectrophotometry and Fluorescence

Absorbance measurements were performed on a Hewlett-Packard 8452Aphotodiode array spectrophotometer. Fluorescence measurements were doneon a Shimadzu RF5000U spectrofluorometer. Protein hydrolysates weredried in a Savant (Savant Instruments, Farmingdale, N.Y.) speed-vacconcentrator. An N-evap (Organomation, Berlin, Mass.) was used to drysamples under nitrogen.

Reactions of Pyridoxamine With PUFAs

PM (1 mM) was reacted with arachidonate, linoleate or oleate (5 mM) at37° C. in 200 mM sodium phosphate buffer, pH 7.4 for 6 days. Aliquotswere removed at days 0, 1, 3 and 6, quenched with 1 mM DTPA and storedat −20° C. until analysis. Modification of the amino group of PM wasdetermined by the trinitrobenzenesulfonic acid (TNBS) assay (Spadaro,1979). Reactions were analyzed by RP-HPLC using a Supelco (Bellefonte,Pa.) C-18 column (25 mm×4.6 cm) with the gradient described in Table 2.3(solvent A=0.1% HFBA, solvent B=acetonitrile).

TABLE 2.3 TIME % A % B FLOW (ml/min) Initial 100 0 1 2 100 0 1 20 40 601 30 40 60 1 31 25 75 1 41 25 75 1 42 100 0 1 55 100 0 1

Products were monitored by fluorescence (Ex=328 nm and Em=393 nm) orabsorbance at 294 nm. For some analyses, pyridoxal was added as aninternal standard prior to injection.

PM-adducts were isolated on a Beckman (Berkeley, Calif.)semi-preparative RP-HPLC column using the gradient described in Table2.4 at a flow of 2 ml/min (solvent A=0.1% HFBA and solventB=acetonitrile).

TABLE 2.4 TIME % A % B FLOW (ml/min) Initial 75 25 2 30 75 25 2 40 70 302 60 70 30 2 61 30 70 2 80 30 70 2 85 75 25 2 100  75 25 2

Reactions of Ribonuclease With Arachidonate in the Presence ofPyridoxamine

RNase (1 mM, 13.7 mg/ml) was reacted with arachidonate (100 mM) alone orin the presence of PM (1 mM) in 200 mM sodium phosphate buffer, pH 7.4.The reactions were done in glass scintillation vials in a 37° C. shakingwater bath for 6 days, and were prepared using sterile technique toprevent bacterial growth. Aliquots were taken at 0, 1, 3 and 6 days andfrozen at −20° C. after addition of 1 mM DTPA/phytic acid to quench allmetal catalyzed oxidation chemistry.

For GC/MS analysis of lipoxidation products, approximately 1 mg ofprotein was delipidated according to the method of Folch et al. (Foich,1957). The lower organic phase was discarded and the aqueous phasereduced with 500 mM NaBH₄ (in 0.1 N NaOH) in 0.1 M borate buffer, pH 9for 4 hours at room temperature. The samples were transferred todialysis tubing (molecular weight cut-off 6,000-8,000) and dialyzedagainst deionized water for 24 hours at 4° C. with 4 water changes. Thedialysates were dried in vacuo, internal standards (d₈-lysine, d₄-CML,d₄- or d₈-CEL, d₈-MDA-lysine and d₄-HNE-lysine) were added, thenhydrolyzed in 6 N HCl at 110° C. for 24 hours following. Thehydrolysates were reconstituted in 1 ml of 0.1% TFA and applied to a1-ml Sep-Pak (Waters Corporation, Milford, Mass.), eluted with 3 ml of0.1% TFA containing 20% methanol, then dried in vacuo. Lysine (FIG. 39)CML, CEL (FIGS. 40 and 41), MDA-lysine and HNE-lysine (FIGS. 42 and 43)were derivatized to their trifluoroacetyl methyl esters for GC/MSanalysis (Knecht, 1991). Methyl esters were prepared by addition of 1 mlof methanolic-HCl (18.7 ml of anhydrous MeOH+1.3 ml acetyl chloride)followed by heating at 65° C. for 1 hour. The methyl esters were driedunder nitrogen then converted to trifluoracetyl derivatives by additionof 1 ml N,O-trifluoroacetic acid anhydride and reaction at roomtemperature for 1 hour. The derivatized samples were dried undernitrogen and reconstituted in 150 μl methylene chloride for GC/MSanalysis.

For GC/MS analysis, the injection port was maintained at 275° C. and thetemperature program was: 4 min at 140° C., 5° C./min ramp to 220° C.,25° C./min ramp to 300° C., then hold at 300° C. for 5 min.Quantification was based on standard curves constructed from mixtures ofdeuterated and non-deuterated standards. Analyses were performed usingselected ion monitoring GC/MS; the following ions were used: lysine and[²H₈]lysine, m/z 180 and 187, respectively; CML and [²H₄]CML, m/z 392and 396; CEL and [²H₄]CEL, m/z 379 and 383; MDA-lysine and[²H₈]MDA-lysine, m/z 474 and 482; HNE-lysine and [²H₄]HNE-lysine, m/z323 and 331.

LDL Oxidations in the Presence of PM

LDL was isolated by density centrifugation, then passed through PD-10(Pharmacia, Sweden) columns pre-equilibrated with 30 ml of PBS (1.5 mlof LDL per column) to remove salts and EDTA, then passed through aCostar 0.4 μm syringe filter (Cambridge, Mass.) to remove aggregates.The filtered LDL (50 μg/ml) was oxidized in PBS with 5 μM Cu²⁺ for 4-5hours. Conjugated diene formation was measured at 294 nm at 10-15 minuteintervals. For measurements of lipoxidation products, 1 mg (20 ml) ofprotein was removed at various time intervals and reduced immediatelywith 500 mM NaBH₄ (added dry weight) in 0.1 M borate buffer pH 9 in thepresence of 1 mM DTPA overnight at 4° C. The reduced samples weredialyzed against dionized (DI) water for 2 days at 4° C. then dried invacuo. The dialysates were reconstituted in 0.5 ml of DI water anddelipidated with MeOH:ether (3:1, v/v). Briefly, 3 ml of MeOH:ether wasadded to the samples with vortexing and the samples were iced for 10minutes to allow protein precipitation. Following centrifugation inrefrigerated centrifuge, the supernatant was removed and discarded. Oneml of ether was added to the pellets, the samples were vortexed, icedfor 10 minutes, centrifuged, and the supernatant discarded. The etherwash was repeated once more.

Following delipidation, deuterated internal standards were added and thesamples were hydrolyzed in 6 N HCl for 24 hours at 110° C. and dried invacuo. The hydrolysates were reconstituted in 1 ml of 1% TFA and appliedto 1 ml Sep-Pak (Waters, Milford, Mass.) cartridges pre-washed andequilibrated with 4 ml of MeOH and 4 ml of 1% TFA. Lipoxidation productswere eluted with 3 ml of 1% TFA in 20% MeOH. After drying in vacuo, thesamples were derivatized to trifluoroacetyl methyl esters and analyzedby GC/MS as described above.

For measurement of lysine modification during lipid peroxidation of LDL,100-150 μg of protein were removed at various time intervals anddelipidated according to the method of Folch (1957). The samples werehydrolyzed in 6 N HCl at 110° C. for 24 hours, dried in vacuo thenanalyzed by cation-exchange HPLC.

Analysis of Thiobarbituric Reactive Substances (TBARs)

PM (1 mM) was reacted with linoleate (5 mM) in 200 mM sodium phosphatebuffer for 6 days at 37° C. Aliquots were removed at 0, 1, 3 and 6 days,quenched with 1 mM DTPA and frozen at −20° C. until analysis. TBARs wereanalyzed according to the method of Sawicki (1963).

Analysis of Linoleate by GC/MS

PM was reacted with linoleate as described above, except each time-pointwas prepared as an independent incubation that was quenched with DTPAand frozen at −20° C. until analysis. Linoleate was extracted directlyfrom the reaction vial according to the method of Folch (1957). Briefly,1 ml of chloroform:methanol (2:1, v/v) was added to the aqueous sample,made 20% in 2N HCl, with vortexing. Following centrifugation, the upperaqueous layer was removed and discarded. Extraction of the organic layerwas repeated once more by adding 1 ml of DI water, vortexing, discardingthe aqueous phase, then drying the organic phase under nitrogen.Palmitate, the internal standard, was added to all samples, followed byderivatization using boron trichloride-methanol in the presence of 1 mMbutylated hydroxy toluene (BHT). Boron trichloride-methanol (0.5 ml) wasadded to the dried linoleate samples and heated at 80° C. for 1 hour.Following derivatization, the samples were dried under nitrogen and thenonpolar methyl esters extracted twice with hexane:water (2:1 v/v). Thehexane layers were pooled and dried under nitrogen. The methyl esterswere reconstituted in 150 μl of methylene chloride then analyzed byGC/MS using single ion monitoring (SIM) of m/z fragments 294 (linoleate)and 220 (palmitate). The temperature program as follows: 150° C., holdfor 3 minutes; 5° C./minute ramp to 180° C.; 8° C./minute ramp to 300°C., hold for 5 minutes. Linoleate was quantified based on standardcurves generated from linoleate and palmitate standards.

Characterization of PM-linoleate Adducts

Synthesis of N-Hexanoyl-pyridoxamine (Product 267)

N-hexanoyl-pyridoxamine was synthesized from pyridoxaminedihydrochloride and hexanoyl (caproyl) chloride. PM (20 mg) wasdissolved in 50 ml of 2N NaOH in a 250 ml round bottomed flask. Hexanoylchloride (in 30 ml ether) was added drop-wise over an hour to the PMsolution; the reaction was kept on ice and stirred vigorously. Followingaddition of hexanoyl chloride, the reaction was allowed to proceed for1-2 hours. The ether layer was then removed and dried under nitrogen.The resulting product appeared as a white residue and was fully solublein water:acetonitrile (50:50 v/v).

Acetylation of PM for GC/MS Analysis

PM was acetylated by reaction at room temperature for 2 hours withacetic anhydride/pyridine (1:1, v/v). The solvent was evaporated invacuo and the acetylated PM was reconstituted in methylene chloride andanalyzed by GC/MS.

Hydrolysis of Product 267

Product 267 was hydrolyzed in 2 N HCl for 4 hours at 95° C. Thehydrolysate was then dried in vacuo. The resulting free hexanoic acidwas analyzed by GC/MS as its propyl ester. Briefly, 1 ml of HCl in drypropanol was added to hexanoic acid and allowed to react at 65° C. for 1hour. Following esterification, the hexanoic acid propyl esters wereextracted with 2 ml hexane:water (2:1, v/v). After vortexing, thesamples were centrifuged and the upper organic layer removed.Concentration of the hexane layer was performed under nitrogen and onice to avoid loss of the volatile propyl esters. The temperature programfor GC/MS analysis was as follows: initial temperature 75° C., 6° C./minramp to 110° C., 10° C./min ramp to 180° C. hold 5 minutes, 12° C./minramp to 270° C./min hold 5 minutes.

The aqueous phase containing PM was dried in vacuo and PM was eitherderivatized and analyzed by GC/MS or analyzed by RP-HPLC as describedabove.

Synthesis of Product 339

Synthesis of product 339 was loosely based on the method of D'Alelio etal. (1937). Briefly, 0.3 grams of PM in 0.3 ml of deionized water wascombined with 0.3 grams of azelaic acid monomethyl ester and heated at140° C. for 10 hours. The brown reaction mixture was then dried in vacuoand reconstituted in deionized water:acetonitrile (1:1, v/v). Thereaction mixture was diluted with 0.1% HFBA and analyzed by LC-ESI-MS.After purification by RP-HPLC, product 339 was hydrolyzed in 2N HCl at95° C. for 4 hours. The hydrolysates were analyzed for free PM andnonanedioic acid. PM was analyzed as described above, azelaic acid wasanalyzed by GC/MS as its dimethyl ester.

Preparation of Hexanoylated-BSA

To BSA (5 mg/ml) in 0.1 M sodium borate buffer, pH 9 was added hexanoylchloride drop-wise over a period of one hour. The reaction was kept onice with vigorous stirring. Following addition of the acyl chloride, thereaction was continued for at least 2 hours. The samples were thendialyzed against DI water for 24 hours at 4° C. with 4 water changes.The dialysates were dried in vacuo and reconstituted in DI water.Protein recovery was determined using the Lowry (1951) assay, and aminogroup modification was determined by the TNBS assay.

Results

Reaction of PM With Linoleate

PM, when reacted alone, or when reacted with oleate (a monounsaturatedfatty acid), remained stable. However, in the presence of arachidonateand linoleate, approximately 60% of PM was consumed by the end of 6days. These results suggested that PM was reacting with products of PUFAoxidation.

To determine if the primary amino group of PM was modified duringreaction with PUFAs, the incubations were analyzed using the TNBS assayas shown in FIG. 44. The amino group of PM was not modified when PM wasincubated alone or in the presence of oleate, the monounsaturatedcontrol. In contrast, approximately 60% of amino groups were modifiedwhen PM was reacted with the PUFAs linoleate and arachidonate. Fromthese results we concluded that the primary amino group of PM wastrapping products of PUFA oxidation.

For the characterization of products, PM was reacted with linoleate for6 days at a molar ratio of 1:5 (linoleate was chosen because it is theprimary fatty acid in LDL). In these experiments we were looking for thetime-dependent formation of new products eluting downstream from PM thatwould represent PM-adducts to oxidized linoleate. Two new products wereobserved that eluted around 24 minutes. Based on fluorescence, the twoproducts accounted for less than 5% of the PM consumed. Considering thatalmost 60% of PM was consumed overall, it was surprising that no otherproducts were observed. To verify that some products were not bindingirreversibly to the reverse-phase C-18 column because of theirhydrophobicity, a total fluorescence scan was taken of the PM/linoleatereaction at time 0 and 6 days. The loss of fluorescence determined bythese scans was identical to the loss of PM fluorescence determined byRP-HPLC, indicating that the loss in fluorescence was real and not afunction of irreversible binding of hydrophobic products to the column.

Because the fluorescent products represented a small fraction of the PMloss, the reactions were analyzed again using absorbance detection at294 nm. The products eluting between 27 and 28 minutes representproducts of linoleate oxidation. However, most of the products elutingbetween 20 and 25 minutes had an extracted absorbance maximum of 294 nm,suggesting that they contained the PM nucleus. The area containing thepotential PM-adducts was collected by semi-preparative RP-HPLC andsubjected to LC-ESI-MS. The extracted mass spectrum of the majorelectrospray active products is shown in FIG. 45. The major products hadm/z values of 267, 305, 479, and 339; 323 was assumed to be the hydratedform of 305. None of these products were present in reactions oflinoleate or PM alone. The kinetics of formation of the 3 most abundantproducts (267, 339 and 305) were determined by RP-HPLC using pyridoxalas an internal standard (FIG. 46). All three compounds increased withtime, with products 267 and 339 consistently forming in the highestyields. For this reason, 267 and 339 were chosen for characterization.

To investigate the chemical properties of 267 and 339, both productswere isolated by RP-HPLC from a semi-preparative column, then hydrolyzed6 N HCl for 24 hours at 110° C., conditions normally used for amino acidanalysis. We later learned that hydrolysis was complete in 4 hours at95° C. Both 267 and 339 were labile to conditions of acid hydrolysis.Two important observations were made from this experiment. First,hydrolysis of the two compounds confirmed that each contained PM. Therecovery of PM was quantitative (based on RP-HPLC analysis usingabsorbance detection), indicating one PM molecule per molecule ofPM-adduct. Additionally, the quantitative recovery of PM allowed us toquantify 267 and 339 using the same extinction coefficient used for PM.Secondly, the linkage between PM and the rest of the compound was acidlabile, indicating that it may be either a Schiff base or amide linkage.To distinguish between the two, both 267 and 339 were reduced withsodium borohydride and analyzed by ESI-MS (direct injection). Reductionof a Schiff base would result in an increase of two molecular weightunits. After reduction, no change in molecular weight was observed,indicating that neither compound was reducible, which eliminated thepresence of a Schiff base linkage. Based on the acid lability andnon-reducible properties of 267 and 339, two amide derivatives of PMwere proposed, and are shown in FIG. 47.

Characterization of Product 267

Our proposed structure for product 267 was a hexamide derivative of PM.The hexamide derivative was prepared synthetically using PM and caproyl(hexanoyl) chloride (see Materials and Methods). The synthetic reactionmixture was analyzed by LC-ESI-MS to confirm formation of thePM-hexamide derivative. The TIC from the LC-MS shows that 267 was themajor product formed during the reaction. The extracted ion chromatogramof m/z 267 showed that only one product having a molecular weight of 267was formed during the reaction. To verify that the primary amino groupof PM participated in an amide linkage (a PM-hexanoate ester derivativewould also have a molecular weight of 267), synthetic product 267 wasisolated by RP-HPLC then analyzed by the TNBS assay. An equivalentamount of PM standard was analyzed in parallel with product 267 forcomparison. No TNBS reactivity was seen in the synthetic 267 compared tothe PM standard, indicating that the primary amino group of PMparticipated in an amide linkage. Next, the synthetic reaction mixturewas analyzed by RP-HPLC and compared to authentic 267 isolated from aPM/linoleate reaction. A mixing experiment between the authentic andsynthetic 267 confirmed that the two compounds co-eluted. Finalstructural verification of 267 (both synthetic and authentic) involvedhydrolysis of the compound into free hexanoic acid and PM, followed byderivatization of each for GC/MS analysis. Hydrolysis of synthetic 267yielded PM and of hexanoic acid in a 1:1 ratio. Identical results wereobtained following hydrolysis of authentic 267 isolated from aPM/linoleate reaction. From the results described above we concludedthat PM-hexamide is one of the major products formed during reaction oflinoleate with PM.

Characterization of Product 339

The proposed structure for product 339 corresponds to a PM-nonanedioicacid-amide derivative. To test this hypothesis, synthetic 339 was madeby reaction of PM with nonanedioic acid monomethyl ester. The reactionmixture was then analyzed by LC-MS, and the resulting extracted ionchromatogram and mass spectrum confirmed the formation of 339.

Synthetic 339 was isolated by RP-HPLC and analyzed by the TNBS assay toverify covalent attachment of the nonanedioic acid to the primary aminogroup of PM. The TNBS reactivity was compared to an equivalent amount ofPM standard and the results are shown in FIG. 48. No TNBS reactivity waspresent in 339, verifying that the amino group of PM participated in theamide bond. For further confirmation of structure, synthetic 339 washydrolyzed in 2N HCl at 95° C. for 4 hours to release free PM andnonanedioic acid. The hydrolysates were derivatized and analyzed byGC/MS for acetylated PM and nonanedioic dimethyl ester. Hydrolysis of339 yielded complete recovery of PM and of nonanedioic acid in a 1:1ratio.

SUMMARY

Modification of the lysine residues of apoB-100, the apoproteinassociated with LDL, is a hypothesized early event in the development ofatherosclerosis. During copper oxidation of LDL, approximately 25% ofthe lysine residues are modified. However, less than 1% of these lysinemodifications have been characterized. The overall purpose of thisresearch project was to identify modifications of lysine residues ofprotein that form during lipid peroxidation reactions. To do this, PMwas used to trap products of linoleate oxidation, in vitro, therebyinhibiting the modification of lysine residues in LDL. From theseexperiments, the two major PM-adducts were identified as N-hexanoyl-PMand N-nonanedioyl-PM. Identification of these two products suggest thatamide derivatives of lysine may represent a significant fraction of thelysine modifications that occur during the oxidation of LDL, both invitro and in vivo. This would explain the observations of Steinbrecher(1987) that a major fraction of lysine modifications in oxidized LDL areresistant to sodium borohydride reduction and are labile to acidhydrolysis.

Discussion

The decrease of free amino groups during reaction of PM with linoleateand arachidonate indicated that PM was trapping products of lipidperoxidation. The amount of TNBS loss agreed well with total PMconsumption determined by RP-HPLC, suggesting that the major interactionbetween PM and PUFAs involves modification of the primary amino group.One of the original reasons for using PM as a trapping agent was toutilize its fluorescence properties for product identification andisolation. Interestingly, the fluorescence response of 267 and 339 isquenched by about a factor of 4 compared to the absorbance response.Fluorescence response also varies among the different B₆ derivatives.For example, pyridoxal is about half as fluorescent as PM, whilepyridoxic acid is twice as fluorescent as PM. From these observations itis apparent that slight variations in the structure of PM causesignificant changes in its fluorescence. For this reason, whenquantifying products 267 and 339 we used absorbance at 294 nm.

As determined by RP-HPLC, of the 50-60% of PM that was consumed duringreaction with linoleate, products 267 and 339 accounted for 10% and 5%of the consumed PM, respectively (product 305 accounts for about 2%).Considering the low levels of other lipoxidation markers, products 267and 339 may constitute major adducts on lipoxidized protein. Only fourlipoxidation products have been characterized to date, and each of thesehas various limitations. For example, CML and CEL are both carbohydrateand lipid derived products, complicating conclusions regarding theorigin of protein damage in vivo. MDA-lysine and HNE-lysine, while bothpurely lipid derived products, are subject to rearrangement to otherproducts, including formation of crosslinks. Furthermore, all fourmarkers represent less than 1% of the lysine modifications formed duringlipoxidation reactions. Consequently, the need for additional markers oflipid-derived damage to proteins is evident, and the identification ofamide derivatives of lysine may provide a convenient and sensitive toolfor assessing protein oxidation. In fact, Kato and colleagues (1999)recently identified the hexamide derivative of N-benzoyl-glycyl-L-lysinefollowing reaction with the 13-hydroperoxide of linoleic acid. Alsosupportive is that approximately half of the lysine modifications formedduring oxidation of LDL are acid labile. The structure of the labileproducts are not known, but hexanoic acid-, nonanedioic acid- or otheramide derivatives of lysine may represent a large portion of such lysinemodifications in vivo. Based on studies of 267 and 339, these types ofderivatives can be hydrolyzed under relatively mild conditions of acidhydrolysis, namely 2N HCl at 95° C. for 2 hours. Once hydrolyzed, thereleased carboxylic acid derivatives could be esterified and measured byGC/MS.

While this aspect of the present invention is not limited to a specificmethod of formation of PM adducts of linoleate oxidation, a proposedmechanism of formation of 267 and 339 is shown in FIG. 49. Thehydroperoxides produced upon oxidation of linoleic acid are known todehydrate to form ketoacids (Spiteller, 1998). Nucleophilic attack onthe keto acid by PM would form a Schiff base, which could then undergooxidative cleavage to form the corresponding amide derivative.Theoretically, oxidative cleavage could occur on either side of theimine carbon. However, formation of the alternate cleavage products wasnot observed during reactions of PM with linoleate. The preference forcleavage and release of the vinyl group is probably driven bystabilization of an intermediate radical by the conjugated hydrocarbonsystem (FIG. 50).

Clearly, lipid peroxidation is an extremely complicated series ofreactions that is sensitive to variations in reaction conditions. Fromour model system of PM and linoleate, we propose that amide derivativesof lysine may constitute a significant fraction of the lysinemodification formed in vivo. Thus, these derivatives can serve asdiagnostic markers for in vivo oxidative protein damage. Preferably, thepresence and/or concentration of amide derivatives of lysine, includingbut not limited to hexanoic acid and nonanedioic acid, are determinedfrom urine, blood, or skin biopsy samples. In a most preferredembodiment, both blood plasma protein and skin biopsy samples are used.The levels of amide derivatives in blood plasma and/or urine serves asan indicator of current exposure, since the half-life of plasma proteinsis approximately 2-3 weeks. Conversely, the half life of skin proteinssuch as collagen and elastin is approximately 35 years, and thusdetermination of amide derivatives of lysine in skin collagen serves asa diagnostic marker for accumulated chemical damage. Such determinationscould utilize the chemical methods detailed herein, or could involvequantitative immunochemical techniques, which would require antibodiesspecific for the derivative form of the protein to be analyzed (forexample, lipoprotein). The immunochemical method (and kits forpracticing the method) could also utilize protein standards, (such asmodified lipoprotein, where the extent of modification is known) andcontrol samples (for example, the appropriate body fluid from someonewith chronic atherosclerosis and/or from someone free ofatherosclerosis.

As an additional note, the trapping properties of PM suggest that it maybe an effective inhibitor of the oxidative modification of lysineresidues on protein during lipid peroxidation reactions.

EXAMPLE 7 Pyridoxamine Inhibits Lipoxidation Reactions, In VitroIntroduction

Clearance of native LDL from the circulation is dependent on recognitionof LDL by the LDL receptor in liver and peripheral tissues. Interactionsbetween this receptor and the apoB-100 component of LDL occur between aregion of acidic amino acids on the receptor and a lysine-rich region onthe protein (Goldstein, 1974). Covalent modification of the lysineresidues (by acetylation, or by reaction with MDA, HNE and other lipidperoxidation products) in the receptor binding region of apoB-100prevents recognition of LDL by the receptor (Steinbrecher, 1987).Consequently, the modified LDL is taken up the scavenger receptor ofmacrophages leading to foam cell formation, a hypothesized initiatingevent in the pathogenesis of atherosclerosis.

Oxidative modification of LDL in vitro interferes with its recognitionby the LDL receptor and promotes its recognition by the macrophagescavenger receptor. Transition metal ions accelerate the oxidation ofLDL, and for this reason, copper-catalyzed oxidation of LDL in vitro isa commonly used model for studying chemical modifications of LDL invivo. Characteristic changes that occur to copper oxidized LDL includeincreased fluorescence (Ex. 350 nm, Em. 433 nm), increased conjugateddiene formation (absorbance at 234 nm), increased anodic electrophoreticmobility, increased uptake by macrophages, and chemical modification ofthe lysine residues of apoB protein (Esterbauer, 1992). The majority oflysine modifications that occur during oxidation of LDL areuncharacterized. However, it is generally accepted that themodifications arise from reaction of carbonyl and dicarbonyl compoundswith the primary, ε-amino group of lysine residues. As a result, anumber of nucleophilic compounds have been evaluated as traps forreactive carbonyls and inhibitors of oxidative modifications of LDL andother proteins.

Experimental Design

The reactive carbonyl and dicarbonyl compounds produced during oxidationof sugars are also produced during oxidation of PUFAs. Because PM was aneffective AGE inhibitor during glycation reactions (presumably bytrapping carbonyls) we hypothesized that PM would also act as aneffective lipoxidation inhibitor.

This example describes the experiments used to test PM's effectivenessas an inhibitor of lipoxidation reactions. We began with a model proteinsystem of RNase incubated with arachidonate in the presence and absenceof PM. Arachidonate was chosen for these studies because of its rapidrate of oxidation (the initial biphasic reaction mixtures becomemonophasic within 2 days). The formation of CML, CEL, MDA-lysine,HNE-lysine and loss of free lysine residues was monitored during theoxidation of arachidonate. In these experiments the RNase concentrationwas 1 mM (10 mM lysine), PM was 1 mM, and arachidonate was 100 mM.

Experiments with model protein were followed by experiments with thebiologically relevant protein, LDL. Copper-catalyzed oxidation of LDLwas monitored by measurements of conjugated diene (A234 nm) formation,changes in electrophoretic mobility, formation of lipoxidation productsand modification of lysine residues.

Results

PM Inhibition of Lipoxidative Modification of RNase by Arachidonate

As shown in FIG. 52, CML, CEL, MDA-lysine and HNE-lysine were formedrapidly in RNase during incubation with arachidonate. MDA-lysine andHNE-lysine are characteristic products of lipoxidation reactions.Formation of CML was consistent with results of Fu et al. (1996) whofirst showed that CML was a product of lipoxidation as well asglycoxidation reactions. The formation of CEL during reaction of RNasewith arachidonate represents the first evidence that CEL, like CML,forms during both glycoxidation and lipoxidation reactions.Interestingly, unlike CML, which is formed from oxidation of botharachidonate (20:4^(Δ5,8,11,14)) and linoleate (18:2^(Δ9,12)), CEL wasformed in only trace amounts from oxidized linoleate compared toarachidonate, indicating that CML and CEL likely form from differentsources in vivo.

Addition of 1 mM PM to the RNase/arachidonate reactions resulted inalmost complete inhibition of formation of CML, CEL (FIG. 52),MDA-lysine and HNE-lysine (FIG. 53). The early plateau phase and declinein MDA-lysine formation resembles that seen during copper oxidation ofLDL (Requena, 1997) and is thought to result from further reaction ofMDA-lysine to form cross links, such as lysine-MDA-lysine (LML)(Requena, 1997).

To examine PM's effect on total lysine modification, we quantifiedlysine by cation-exchange HPLC after hydrolysis of the protein.Lipoxidation products such as MDA-lysine and HNE-lysine contain reactiveSchiff base and aldehyde functional groups that are not stable toconditions of acid hydrolysis. To stabilize these adducts, the proteinwas first reduced with NaBH₄, converting imines and aldehydes to aminesand alcohols, respectively. Amino acid analysis indicated that 58% oflysine residues were modified during reaction of RNase witharachidonate. Lysine loss decreased by approximately one-half innon-reduced samples (data not shown), confirming formation ofacid-labile lysine modifications. PM, at 1 mM, decreased lysine loss toapproximately 5% at the end of 6 days. It is interesting to note thatless than 1% of the 58% modified lysine residues can be accounted for byCML, CEL, MDA-lysine and HNE-lysine.

The measurements of lipoxidation product formation and of free lysinemodification discussed above showed that levels of lipoxidation productsand of lysine modification began to rise at about 3 days. This led us tosuspect that PM was becoming saturated after 3 days of reaction. To testthis, we quantified PM consumption during reactions of RNase with AA byRP-HPLC, which showed that virtually all the PM was consumed after 3days of reaction with AA, explaining the increase in CML, CEL,MDA-lysine, HNE-lysine, and lysine modification after 3 days.

Based on the results obtained from reaction of RNase with arachidonate,we confirmed our hypothesis that PM was a potent inhibitor oflipoxidative modification of proteins, even when present at a 10-foldlower concentration than lysine (in RNase) and a 100-fold lowerconcentration than PUFA. During reaction of RNase with AA, the reactionmixture changed from biphasic to monophasic after 2 days. The inclusionof PM during reaction of RNase with AA appeared to prolong the changefrom a biphasic to a monophasic system to approximately 3-4 days, but by6 days the PM-containing reactions were also monophasic, indicatingcomplete oxidation of AA. The fact that PM allowed oxidation of AA butinhibited modification of RNase indicated that PM's primary mechanism ofaction involved trapping of intermediates produced during the lipidperoxidation reactions.

PM Inhibits Lipoxidative Modification of LDL During Metal-catalyzedOxidation

Since PM inhibited lipoxidation chemistry in the RNase/arachidonatemodel protein system, we evaluated its effectiveness as an inhibitor oflipoxidation during copper-catalyzed oxidation of LDL. Because oflimited LDL protein availability, the LDL experiments were done in twoparts. The first set of LDL oxidations was done in the presence of 10 or100 μM PM, and the effect of PM on CML, CEL, MDA-lysine and HNE-lysineformation was measured by GC/MS. For the second set, the concentrationof PM was increased to 100 and 250 μM, and the effects of PM onconjugated diene formation, electrophoretic mobility, and lysinemodification were determined.

At concentrations of 100 and 250 μM PM, in a concentration-dependentmanner, prolonged the lag phase of conjugated diene formation duringoxidation of LDL (FIG. 54) and lowered the total amount of conjugateddienes present at the end of the oxidation. As with RNase, oxidation ofLDL proceeded in the presence of PM, yielding similar amounts ofA_(234 nm). PM, at both concentrations, also decreased theelectrophoretic mobility of oxidized LDL in comparison to the copperoxidized control. Copper oxidation of LDL resulted in formation of CMLat levels consistent with those previously reported by Requena et al(1997). Similar to the results obtained during reaction of RNase witharachidonate, CEL was formed at concentrations approximately 10-foldlower than CML. PM, at 100 and 250 μM, inhibited CML (70% and 80%,respectively) CEL (43% and 71%) (FIG. 55), MDA-lysine (8% and 46%) andHNE-lysine (60% and 83%) formation (FIG. 56). In addition, total lysinemodification was decreased by 62% by 100 μM PM and 87% by 250 μM PM innonreduced and reduced samples (FIG. 57). CML, CEL, MDA-lysine andHNE-lysine accounted for approximately 1% of the lysine modifications.These results demonstrate that PM not only inhibits formation of CML,CEL, MDA-lysine and HNE-lysine, but also provides protection againsttotal lysine modification during copper-catalyzed oxidation of LDL.

PM has Minimal Antioxidant Activity Against Lipid Peroxidation

The TBARs assay recognizes primarily malondialdehyde, but also reactswith other compounds produced during PUFA oxidation (such as glyoxal).It is likely that some TBA-reactive compounds continue on to form otherproducts, as shown in FIG. 58. For example, aldehydes that maypolymerize via aldol condensation reactions. This would explain thedecrease in TBARs. The production of TBARs during oxidation of linoleateincreased during the first 3 days of reaction then decreased by day 6(FIG. 58). PM slowed the rate of formation of TBARs by approximately30%, but did not have a significant effect on the final yield of TBARsin the reaction mixture. Overall, the inhibitory effect of PM on therate of TBARs formation during oxidation of linoleate suggests that PMdoes have some antioxidant activity, retarding, rather than completelyinhibiting, lipid peroxidation.

Oxidation of linoleate (5 mM) in phosphate buffer for 6 days resulted incomplete destruction of the PUFA by 3 days. The rate of linoleateoxidation was slowed approximately 10% by 1 mM PM, though the finalextent of linoleate oxidation was not affected (FIG. 59). The results ofthese experiments are consistent with those on TBARs formation and LDLoxidation, in that PM slowed the kinetics of lipid peroxidation but hadlittle effect on the final extent of oxidation.

Discussion

The trapping of lipid intermediates by PM suggested that it mightinhibit the modification of proteins by lipid peroxidation reactions byacting as a sacrificial nucleophile. To test this hypothesis, PM wasincluded in reactions of model protein (RNase) with arachidonic acid.The effect of PM on copper-catalyzed oxidation of LDL was also studied.In both the RNase/arachidonate system and during oxidation of LDL, PMinhibited the formation of the lipoxidation products CML, CEL,MDA-lysine and HNE-lysine. PM also prevented the modification of lysineresidues during these reactions. The results of these experimentssuggest that PM might be useful therapeutically for inhibiting theoxidative chemical modification of proteins and thereby limiting theprogression of chronic diseases such as diabetes and atherosclerosis.

Exposure of RNase to a 100-fold excess of peroxidizing arachidonate overa period of 6 days resulted in rapid oxidation and solubilization(within 2 days) of the arachidonate and a high percentage of lysinemodification. Visually, reactions containing PM remained biphasic longerthan those without, indicating that PM had some chelating or antioxidantactivity. These visual observations were confirmed chemically when PMwas shown to inhibit formation of CML, CEL, MDA-lysine and HNE-lysine,despite the large excess of PUFA to PM (100:1) and of lysine to PM(10:1).

The efficiency with which PM protected against total lysinemodifications despite the excess of PUFA, further established that PM isan effective inhibitor of lipoxidation reactions. The concentration ofreactive carbonyls produced during oxidation of 100 mM arachidonategreatly exceeds the trapping capacity of 1 mM PM (assuming 1 carbonyltrapped per molecule PM), yet PM still was an effective protector oflysine residues. This suggests that only a small fraction of lipidperoxidation products may participate in lysine modification.Alternatively, PM may intercept early lipid peroxidation products,preventing further oxidation of the products into a larger number ofshort-chain carbonyl compounds that could also modify protein. This isconsistent with the observation that PUFA reactions containing PMappeared to become soluble (monophasic) more slowly than PUFAs oxidizedalone. The PM-containing reactions probably contain more long chain,insoluble compounds (presumably covalently attached to PM) in comparisonto the reactions without PM. During lipid peroxidation, it is possiblethat the majority of protein modification occurs by reaction of shortchain, water-soluble carbonyl compounds with primary amino groupsexposed to the aqueous environment. By preventing the formation ofshort-chain lipid peroxidation products, PM would protect the proteinagainst “carbonyl stress” and therefore would undergo less modification.The result: an effective degree of protein protection by a minimumconcentration of PM, an attractive quality for a potential drug.

The inhibition of lipoxidation in the RNase model system suggested thatPM would have similar effects during copper-catalyzed oxidation of LDL.As shown in FIG. 54, both 100 and 250 μM PM, in a dose-dependent manner,were effective in prolonging the lag phase and lowering the total amountof conjugated dienes formed. If PM was solely a carbonyl trap, it shouldhave no effect on the initiation phase of lipid peroxidation, but wouldbecome effective during the propagation phase, when the hydroperoxidesbreak down into smaller products. However, the fact that PM prolongs thelag phase of conjugated diene formation suggests that PM has someradical scavenging or chelating activity. In fact, the structure of PMis not unlike many compounds that function as radical scavengers; mostaromatic amines or phenols have antioxidant activity (Halliwell, 1989).It is possible that PM, like Vitamin E, reacts with lipid peroxy andalkoxy radicals, thus slowing the propagation phase of lipidperoxidation.

The increase in electrophoretic mobility observed following LDLoxidation is poorly understood. Several processes are probablyresponsible for the increase in negative charge. The most logicalexplanation is modification of positively charged amino groups by eitherSchiff's base or Michael adduct formation. An additional possibilityinvolves attachment of negatively charged entities to the surface ofLDL. Some studies suggest the presence of negatively charged groupsformed after copper oxidation of LDL (Brnjas-Kraljevic, 1991). Whateverthe cause, PM, at both concentrations, successfully decreased theelectrophoretic mobility of copper oxidized LDL. Additionally, andsimilar to the results seen in the model experiments, PM effectivelyinhibited CML, CEL, MDA-lysine and HNE-lysine formation during LDLoxidation, in a dose dependent manner. Finally, and perhaps mostimportantly, PM protected against total lysine modification duringoxidation of LDL. Though PM clearly prevented lipoxidation, levels ofeach lipoxidation product appeared to begin increasing afterapproximately 2 hours of oxidation, suggesting consumption of PM duringlater stages of LDL oxidation. This is not unlikely considering theratio of PM to reactive intermediates produced during oxidation of 50μg/ml LDL. The protein concentration of LDL used in our experiments (50mg/ml) corresponds to a PUFA concentration of 117 μM, and a ratio of PMto linoleate and to arachidonate of 1:1 and 8:1, respectively.Esterbauer (1992) reports that oxidation of a similar concentration ofLDL with 1.66 μM copper results in formation of 27 μM total aldehydes,50 μM peroxides, and 24 μM conjugated dienes. Taken together, theconcentration of reactive compounds following oxidation of LDL with 1.66μM copper is roughly 100 μM. Our experiments contained 3 times as muchcopper (5 μM) and we would therefore expect a faster rate of oxidation.Esterbauer's estimation also did not include other reactiveintermediates such as endo- and exo-peroxides, epoxides, ketoacids andsmaller compounds like glyoxal. Nonetheless, using Esterbauer'sestimations, the ratio of PM to reactive intermediates in the LDLoxidations analyzed for CML, CEL, MDA-lysine and HNE-lysine was 1:10 (10μM PM: 100 μM reactive products) and 1:1. Thus, the observation that PMappeared to become saturated (less effective) during the later stages ofLDL oxidation is understandable. However, considering the dose-dependentresponse to PM, it is certain that larger doses of PM would prove moreeffective in preventing protein modification. In addition, the rate ofcarbonyl production during in vitro copper-catalyzed oxidation of LDL ismuch faster than what would occur in vivo. Also encouraging is that μMconcentrations of PM were effective yet nontoxic in a small animal model(as shown above), indicating that larger doses should be tolerable invivo.

That PM retarded but did not prevent lipid peroxidation (as demonstratedby measurement of TBARs formation and rate of linoleate oxidation)suggests that the antioxidant activity of PM is a minor contributor toits mechanism of action. The mild antioxidant effects of PM were notcompletely unexpected. In fact, PM contains two functional groups commonto many antioxidants; most antioxidants are aromatic amines or phenols.These types of antioxidants inhibit the propagation phase of lipidperoxidation by donation of a hydrogen atom to a peroxy or alkoxyradical. The resulting antioxidant radical delocalizes into the aromaticring structure and is not reactive enough itself to perform hydrogenabstraction and further propagate the reaction. FIG. 60 represents apossible mechanism for the antioxidant activity of PM. The ratio of PMto Cu²⁺ in the LDL oxidation experiments was 50:1, yet oxidation of LDLstill took place. From the results shown herein it is clear that themajor mechanism of inhibition of PM, at least during reaction withlinoleate, probably does not involve radical scavenging. For example,during oxidation of LDL, PM slowed conjugated diene formation (an indexof PUFA oxidation) but did not prevent it. In contrast, PM virtuallyprevented lysine modification in the same experiment. This implies thatPM acted primarily as a trapping agent (by protecting lysine frommodification) and secondly as an antioxidant (by slowing, but notpreventing, lipid peroxidation).

The finding that PM, in vitro, appears to interrupt lipid peroxidationvia two mechanisms (carbonyl trapping and antioxidant) is encouraging.PM may prove to be a powerful and multifunctional drug for the treatmentof diabetes, atherosclerosis, and other chronic and inflammatorydiseases in which chronic oxidative modifications of proteins isconsidered to be part of the pathogenic process.

Identification of Amide-derivatives of PM: Implication for LysineModifications In Vivo.

During copper-catalyzed oxidation of LDL, approximately half of thelysine modifications that form are labile to conditions of acidhydrolysis (Steinbrecher, 1987). The identification of N-hexanoyl-PM andN-nonanedioyl-PM from reactions of PM with linoleic acid suggests thatamide derivatives may constitute a significant portion of theacid-labile lysine modifications formed during LDL oxidation. Formationof amide derivatives of lysine would also contribute to the increasedelectrophoretic mobility that occurs during LDL oxidation. Kato andcolleagues (1999) have identified the hexamide derivative ofhippuryllysine as a product formed during incubation of hippuryllysinewith the hydroperoxide of linoleic acid. They went on to showimmunohistochemical evidence for the existence of N-hexanoyl-lysine inhuman atherosclerotic lesions.

Linoleate is the major PUFA in LDL; therefore, the formation ofN-hexanoyl- and N-nonanedioyl-lysine would be expected during LDLoxidation based on our results from reactions of linoleate with PM.However, LDL also contains arachidonate, albeit at a much lowerconcentration than linoleate (the ratio of arachidonate to linoleate inLDL is approximately 1:8) (Esterbauer, 1992). However, the oxidation ofAA is more facile than that of linoleate because of the presence of 3bis-allylic carbons, but the formation of amide derivatives may bepossible from this PUFA as well as from linoleate. Like linoleate,arachidonate is an ω-6 PUFA, and produces a 15-hydroperoxide that,through the mechanism we proposed earlier for formation of 267(nucleophilic addition of a primary amine on a ketoacid), couldconceivably lead to formation of N-hexanoyl derivatives of lysine.However, oxidation of arachidonate forms several other hydroperoxidesthat may lead to several amide derivatives of varying chain lengths,such as a pentadioic-amide derivative (formed from the 5-hydroperoxide)and longer chain unsaturated fatty acid derivatives. As a result, futurestudies of amide formation during the oxidation of LDL or from othermodified proteins should not be limited to 6- and 9-carbon chain-lengthacids.

Because amide derivatives are not reducible by sodium borohydride andare not stable to acid hydrolysis, their detection in lipoxidativelymodified proteins would be indirect. Modified protein, such as LDL,could be hydrolyzed under relatively mild conditions, e.g. 2N HCl at 95°C. for 2 hours. This would release the amide derivatives as the freecarboxylic acids, which in turn could be separated from the protein (byorganic extraction) and analyzed as their ester derivatives by GC/MS.Carboxylic acids containing less than 6 carbons may require eitherheadspace GC analysis or conversion to heavy ester derivatives (such asbutyl esters) because of their volatility.

Immunological methods for detection of protein modifications are analternative approach. For example, antibodies could be raised againsthexanoyl-modified proteins then used to detect related derivatives intissues. Production of both polyclonal and monoclonal antibodies is wellknown in the art. See, Antibodies: A Laboratory Manual, Harlow et al.,eds., Cold Spring Harbor, N.Y. (1988). For polyclonal antibodies, ahexanoyl-modified protein(s), in the presence of an adjuvant, isinjected into rabbits with a series of booster shots in a prescribedschedule optimal for high titers of antibody in serum. An extensivedescription for producing monoclonal antibodies derived from the spleenB cells of an immunized mouse and an immortalized myeloma cell is foundin the above reference for polyclonal antisera production. Mice areimmunized with hexanoyl-modified protein(s). Following cell fusion,selection for hybrid cells' and subcloning, hybridomas are screened fora positive antibody against the hexanoyl-modified protein(s) using anindirect ELISA assay Regardless of the method of analysis, theidentification of N-hexanoyl and N-nonanedioyl derivatives of lysine invivo should prove invaluable. For the first time, a significant markerof lipid-derived modification of protein has been identified that shouldbe useful 1) to assess levels of oxidative damage to protein in vivo and2) to better understand the processes that lead to the development ofatherosclerosis.

Inhibition of Lipoxidation by PM: Implications for Drug Therapy

Diabetes is associated with an increased risk of cardiovascular diseaseand is also accompanied by increased lipid peroxidation that mayactually be catalyzed by hyperglycemia (Chisholm, 1992 and Tsai, 1994).The modification of proteins by lipid peroxidation reactions isimplicated in the pathology of various complications associated withdiabetes and atherosclerosis. Specific modifications of protein thathave been identified on lysine residues that occur during lipidperoxidation include, CML, CEL, MDA-lysine and HNE-lysine. Theseproducts represent a very small fraction of the lysine modificationsthat occur during oxidation of LDL, but are the only proteinmodifications currently characterized. Inhibition of these 4 compounds,and inhibition of total lysine modification, by PM in vitro suggeststhat this B₆ vitamer may provide a useful therapeutic agent for thetreatment of complications arising from the chemical modification ofproteins in vivo.

From our experiments with both model protein and copper-catalyzedoxidation of LDL, PM appears to effectively inhibit lipoxidation bytrapping products of lipid peroxidation, although the process of lipidperoxidation itself continues. It is interesting to note that, unlikePM, aminoguanidine (AG), a hydrazine derivative that is also thought toact as a trapping agent, is a potent inhibitor of copper-catalyzedoxidation of LDL (i.e. prevents conjugated diene formation)(Philis-Tsimikas, 1995), though the products formed during AG inhibitionof LDL oxidation have not been identified. Although both PM and AG areconsidered trapping agents, they appear to affect lipid peroxidation atdifferent stages. The prevention of LDL oxidation by AG suggests that itmay have chelating activity. PM, on the other hand, does not behave asif it is chelating metal ions, since it slows the lag phase, but doesnot prevent, conjugated diene formation. It is also possible that,during LDL oxidation, PM works through multiple mechanisms, for example,by trapping intermediates and, to a lesser extent, by scavengingradicals.

PM will likely function through multiple mechanisms in vivo as well. Ourstudies have shown that PM should prevent lipoxidative damage toprotein, thereby impairing the development of atherosclerosis. Previousexamples above have demonstrated that PM inhibits both AGE formationfrom glucose and AGEs from post-Amadori reactions. These findingssuggest that PM may also have inhibitory effects on damage to proteinsderived from carbohydrates. By inhibiting protein damage occurring fromboth lipid and carbohydrate sources, PM would be expected to inhibit thedevelopment of complications in both atherosclerotic and diabetic animalmodels.

EXAMPLE 8 Compounds for Inhibiting Oxidative Protein Modification

The present invention encompasses compounds, and pharmaceuticalcompositions containing compounds having the general formula:

Formula I

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group;

and salts thereof.

The present invention also encompasses compounds of the general formula

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group;

R₄ is H, or C 1-18 alkyl;

R₅ and R₆ are H, C 1-18 alkyl, alkoxy or alkane; and salts thereof.

In addition, the instant invention also envisions compounds of theformulas

The compounds of the present invention can embody one or more electronwithdrawing groups, such as and not limited to —NH₂, —NHR, —NR₂, —OH,—OCH₃, —OCR, and —NH—COCH₃ where R is C 1-18 alkyl.

By “alkyl” and “lower alkyl” in the present invention is meant straightor branched chain alkyl groups having from 1-18 carbon atoms, such as,for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl,3-hexyl, and 3-methylpentyl. Unless indicated otherwise, the alkyl groupsubstituents herein are optionally substituted with at least one groupindependently selected from hydroxy, mono- or dialkyl amino, phenyl orpyridyl.

By “alkoxy” and “lower alkoxy” in the present invention is meantstraight or branched chain alkoxy groups having 1-18 carbon atoms, suchas, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy, neopentoxy,hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

By “alkene” and “lower alkene” in the present invention is meantstraight and branched chain alkene groups having 1-18 carbon atoms, suchas, for example, ethlene, propylene, 1-butene, 1-pentene, 1-hexene, cisand trans 2-butene or 2-pentene, isobutylene, 3-methyl-1-butene,2-methyl-2-butene, and 2,3-dimethyl-2-butene.

By “salts thereof” in the present invention is meant compounds of thepresent invention as salts and metal complexes with said compounds, suchas with, and not limited to, Al, Zn, Mg, Cu, and Fe.

One of ordinary skill in the art will be able to make compounds of thepresent invention using standard methods and techniques.

The instant invention encompasses pharmaceutical compositions whichcomprise one or more of the compounds of the present invention, or saltsthereof, in a suitable carrier. The instant invention encompassesmethods for administering pharmaceuticals of the present invention fortherapeutic intervention of pathologies which are related to AGEformation in vivo. In one preferred embodiment of the present inventionthe AGE related pathology to be treated is related to diabeticnephropathy.

The instant invention may be embodied in other forms or carried out inother ways without departing from the spirit or essentialcharacteristics thereof. The present disclosure and enumerated examplesare therefore to be considered as in all respects illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims, and all equivalency are intended to be embraced therein. One ofordinary skill in the art would be able to recognize equivalentembodiments of the instant invention, and be able to practice suchembodiments using the teaching of the instant disclosure and onlyroutine experimentation.

We claim:
 1. A method for inhibiting oxidative modification of proteinsin a non-hyperglycemic mammal, comprising administering to thenon-hyperglycemic mammal an amount effective to inhibit proteinoxidative modifications of a compound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; R₂ and R₆is H, OH, SH, NH₂, C 1-18 alkyl, alkoxy or alkene; R₄ and R₅ are H, C1-18 alkyl, alkoxy or alkene; Y is N or C, such that when Y is N R₃ isnothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.
 2. A method for inhibiting oxidativemodification of low density lipoproteins in a non-hyperglycemic mammal,comprising administering to the non-hyperglycemic mammal an amounteffective to inhibit low density lipoprotein oxidative modifications ofa compound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; R₂ and R₆is H, OH, SH, NH₂, C 1-18 alkyl, alkoxy or alkene; R₄ and R₅ are H, C1-18 alkyl, alkoxy or alkene; Y is N or C, such that when Y is N R₃ isnothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.
 3. A method for inhibiting lipid peroxidationin a non-hyperglycemic mammal, comprising administering to thenon-hyperglycemic mammal an amount effective to inhibit lipidperoxidation of a compound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; R₂ and R₆is H, OH, SH, NH₂, C 1-18 alkyl, alkoxy or alkene; R₄ and R₅ are H, C1-18 alkyl, alkoxy or alkene; Y is N or C, such that when Y is N R₃ isnothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.
 4. A method for preventing or treatingatherosclerosis, comprising administering to a non-hyperglycemic mammalin need thereof an amount effective to prevent or treat atherosclerosisof a compound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; whereinwhen R₂ is H, OH, SH, NH₂, C 1-18 alkyl, alkoxy or alkene; then R₆ is H,SH, NH₂, C 1-18 alkyl, alkoxy or alkene; or when R₂ is H, OH, SH, NH₂, C1-18 alkoxy then R₆ is H, OH SH, NH₂, C 1-18 alkyl, alkoxy or alkene; R₄and R₅ are H, C 1-18 alkyl, alkoxy or alkene; Y is N or C, such thatwhen Y is N R₃ is nothing, and when Y is C, R₃ is NO₂ or anotherelectron withdrawing group, or salts thereof.
 5. A method for inhibitingthe oxidative modification of proteins in a non-hyperglycemic mammal,comprising administering to the non-hyperglycemic mammal an effectiveamount of pyridoxamine to inhibit the oxidative modification ofproteins.
 6. A method for inhibiting the oxidative modification of lowdensity lipoproteins in a non-hyperglycemic mammal, comprisingadministering to the non-hyperglycemic mammal an effective amount ofpyridoxamine to inhibit the oxidative modification of low densitylipoproteins.
 7. A method for inhibiting lipid peroxidation in anon-hyperglycemic mammal, comprising administering to thenon-hyperglycemic mammal an effective amount of pyridoxamine to inhibitlipid peroxidation.